Antisense modulation of vegf co-regulated chemokine-1 expression

ABSTRACT

Antisense compounds, compositions, and methods are provided for modulating the expression of VEGF Co-regulated chemokine-1 (VCC-1). The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding VCC-1. Methods of using these compounds for modulation of VCC-1 expression and for treatment of diseases associated with expression of VCC-1 are provided.

The present application claims priority under Title 35, United StatesCode, § 119 to U.S. Provisional application Ser. No. 60/404,484, filedAug. 19, 2002, which is incorporated by reference in its entirety as ifwritten herein.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of VEGF Co-regulated chemokine-1 (VCC-1). In particular,this invention relates to antisense compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodingVEGF Co-regulated chemokine-1. Such oligonucleotides have been shown tomodulate the expression of VEGF Co-regulated chemokine-1.

BACKGROUND OF THE INVENTION

Angiogenesis is the growth of new capillary blood vessels frompre-existing vessels and capillaries and is crucial in a large number ofprocesses, such as wound repair, embryonic development, and the growthof solid tumors. In neovascularization, endothelial cells will undergomigration, elongation, proliferation, and orientation leading to lumenformation, re-establishment of a basement membrane and eventualanastomosis with other vessels (Patan, S., 2000 J. Neurooncol. 50(1-2):1-15).

Cytokines are small proteins that bind to cell surface receptors inorder to modulate activity of a variety of cells. VCC-1 appears to be aCXC chemokine, which is a sub-family of the cytokines, named due totheir conserved Cys-Xaa-Cys sequence near the N-terminus of the protein.Family members also contain two additional conserved cysteine residuesand are roughly 70-130 amino acids in size. They are secreted proteinswith a leader sequence of 20-25 amino acids, which is cleaved off beforerelease. A characteristic three-dimensional folding of the chemokines isstabilized by the disulfide bonds that form between the conservedcysteine 1 and cysteine 2 and between cysteine 3 and cysteine 4(reviewed in Baggiolini, M., 2001 J. Int. Med. 250: 91-104).

Among the known CXC chemokines are interleukin-8 (IL-8),γ-interferon-inducible protein 10 (IP-10), platelet factor 4 (PF4),monokine induced by γ-interferon (MIG), epithelial neutrophil activatingprotein-78 (ENA-78), the growth related oncogene peptides (GRO) GRO-α,GRO-β and GRO-γ, and others. These proteins mediate a diverse number ofactivities including activation of neutrophils, induction of chemotaxis,induction of angiogenesis and tumorigenesis, as well as inhibition ofangiogenesis and tumorigenesis (Belperio, J. A., et al., 2000 J. Leuk.Bio. 68: 1-8).

All of the biological effects of chemokines are exerted through theirinteraction with a cell surface receptor. There are six CXC chemokinereceptors (CXCRS) identified to date (reviewed by Horuk et al., 2001Cytokine Growth Factor Rev. 12: 313-335). The CXCRs are members of thesuperfamily of serpentine proteins that signal through heterotrimericG-proteins. These proteins have been shown to possess the ability tobind multiple chemokines with high affinity.

The regulation of angiogenesis is controlled at least in part byangiostatic and angiogenic cytokines. IL-8 has been shown to mediateendothelial cell chemotactic and proliferative activity in vitro(Strieter R. M., et al., 1992, Am. J. Pathol. 141: 1279-1284 and Koch,A. E., et al., 1992 Science 258:1798-1801). In contrast, IP-10, MIG, andPF4 have been found to have angiostatic properties both in vitro and invivo (Maione, T. E., et al., 1990, Science 247: 77-79; Strieter, R. M.,et al., 1995, Biochem. Biophys. Res. Commun. 210(1): 51-57; andArenberg, D A, et al., 1997 Methods Enzymol 283: 190-220).

Since tumor growth is dependent upon angiogenesis, it follows that CXCchemokines play a role in growth and metastasis of tumors. The clearestexample of angiogenic chemokines modulating tumorigenesis and growth wasshown by over-expression of GRO α, β and γ in human melanocytes, whichlead to an anchorage-independent growth phenotype in vitro and theability to form tumors in vivo in nude mice (Luan, J., et al., 1997, J.Leukoc. Bio. 62: 588-597 and Owen, J. D., et al., 1997 Int. J. Cancer73: 94-103). Furthermore, both IL-8 and ENA-78 expression in non-smallcell lung carcinoma (NSCLC) has been correlated with tumor angiogenesis(Yatsunami, J., et al., 1997, Cancer Lett. 120: 101-108, and Arenberg, DA, et al., 1998 J. Clin. Invest. 102: 465-472).

Other CXC chemokines appear to either inhibit tumor cell growth orinduce necrosis of tumor cells. Nude mice with Burkitt's tumorsubcutaneously implanted were inoculated daily with recombinant MIG.This consistently caused tumor necrosis with vascular damage (Sgadari,C., et al., 1997 Blood 89(8): 2635-). The same was seen in Burkitt'stumor bearing nude mice treated with IP-10 (Sgadari, C., et al., 1996Proc. Natl. Acad. Sci. U.S.A. 93:13791-13796). SCID mice bearing NSCLCtumors and treated with MIG also show growth inhibition, decreasednumbers of metastasis, and a decrease in tumor-derived vessel density(Addison, C. L., et al., 2000 Hum. Gene Ther. 11: 247-261).

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of VCC-1 expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to a nucleic acid encoding VCC-1,and which modulate the expression of VCC-1. Pharmaceutical and othercompositions comprising the antisense compounds of the invention arealso provided. Further provided are methods of modulating the expressionof VCC-1 in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of VCC-1 by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cDNA sequence and the VCC-1 protein sequence encodedtherefrom.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding VCC-1, ultimately modulating the amountof VCC-1 produced. This is accomplished by providing antisensecompounds, which specifically hybridize with one or more nucleic acidsencoding VCC-1. As used herein, the terms “target nucleic acid” and“nucleic acid encoding VCC-1” encompass DNA encoding VCC-1, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds, which specifically hybridize to it, isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of VCC-1.In the context of the present invention, “modulation” means either anincrease (stimulation) or a decrease (inhibition) in the expression of agene. In the context of the present invention, inhibition is thepreferred form of modulation, of gene expression and mRNA is a preferredtarget.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding VCC-1. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding VCC-1, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e. 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region “refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region “refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases, whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans. In the context of this invention, the term“oligonucleotide” refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases.

VCC-1 antisense oligonucleotides that have activity in thecardiovascular, angiogenic, and endothelial assays described herein,and/or whose gene product has been found to be localized to thecardiovascular system, is likely to have therapeutic uses in a varietyof cardiovascular, endothelial, and angiogenic disorders, includingsystemic disorders that affect vessels, such as diabetes mellitus. Itstherapeutic utility could include diseases of the arteries, capillaries,veins, and/or lymphatics. Examples of treatments hereunder includetreating muscle wasting disease, treating osteoporosis, aiding inimplant fixation to stimulate the growth of cells around the implant andtherefore facilitate its attachment to its intended site, increasing IGFstability in tissues or in serum, if applicable, and increasing bindingto the IGF receptor (since IGF has been shown in vitro to enhance humanmarrow erythroid and granulocytic progenitor cell growth).

VCC-1 antisense oligonucleotides can be used to inhibit the productionof excess connective tissue during wound healing or pulmonary fibrosisif VCC-1 promotes such production. This would include treatment of acutemyocardial infarction and heart failure.

Moreover, the present invention provides the treatment of cardiachypertrophy, regardless of the underlying cause, by administering atherapeutically effective dose of VCC-1 antisense oligonucleotides.

The treatment for cardiac hypertrophy can be performed at any of itsvarious stages, which may result from a variety of diverse pathologicconditions, including myocardial infarction, hypertension, hypertrophiccardiomyopathy, and valvular regurgitation. The treatment extends to allstages of the progression of cardiac hypertrophy, with or withoutstructural damage of the heart muscle, regardless of the underlyingcardiac disorder.

VCC-1 antisense oligonucleotides would be useful for treatment ofdisorders where it is desired to limit or prevent angiogenesis. Examplesof such disorders include vascular tumors such as hemangioma, tumorangiogenesis, neovascularization in the retina, choroid, or cornea,associated with diabetic retinopathy or premature infant retinopathy ormacular degeneration and proliferative vitreoretinopathy, rheumatoidarthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation,psoriasis, endometriosis associated with neovascularization, restenosissubsequent to balloon angioplasty, sear tissue overproduction, forexample, that seen in a keloid that forms after surgery, fibrosis aftermyocardial infarction, or fibrotic lesions associated with pulmonaryfibrosis.

Specific types of diseases are described below, where VCC-1 antisenseoligonucleotides may serve as useful for vascular-related drug targetingor as therapeutic targets for the treatment or prevention of thedisorders.

Atherosclerosis is a disease characterized by accumulation of plaques ofintimal thickening in arteries, due to accumulation of lipids,proliferation of smooth muscle cells, and formation of fibrous tissuewithin the arterial wall. The disease can affect large, medium, andsmall arteries in any organ. Changes in endothelial and vascular smoothmuscle cell function are known to play an important role in modulatingthe accumulation and regression of these plaques.

Hypertension is characterized by raised vascular pressure in thesystemic arterial, pulmonary arterial, or portal venous systems.Elevated pressure may result from or result in impaired endothelialfunction and/or vascular disease.

Inflammatory vasculitides include giant cell arteritis, Takayasu'sarteritis, polyarteritis nodosa (including the microangiopathic form),Kawasaki's disease, microscopic polyarightis, Wegener's granulomatosis,and a variety 101 of infectious-related vascular disorders (includingHenoch-Schonlein Prupura). Altered endothelial cell function has beenshown to be important in these diseases. Reynaud's disease and Reynaud'sphenomenon are characterized by intermittent abnormal impairment of thecirculation through the extremities on exposure to cold. Alteredendothelial cell function has been shown to be important in thisdisease.

Aneurysms are saccular or fusiform dilatations of the arterial or venoustree that are associated with altered endothelial cell and/or vascularsmooth muscle cells.

Arterial restenosis (restenosis of the arterial wall) may occurfollowing angioplasty as a result of alteration in the function andproliferation of endothelial and vascular smooth muscle cells.

Thrombophlebitis and lymphangitis are inflammatory disorders of veinsand lymphatics, respectively, that may result from, and/or in, alteredendothelial cell function. Similarly, lymphedema is a conditioninvolving impaired lymphatic vessels resulting from endothelial cellfunction.

The family of benign and malignant vascular tumors is characterized byabnormal proliferation and growth of cellular elements of the vascularsystem. For example, lymphangiomas are benign tumors of the lymphaticsystem that are congenital, often cystic, malformations of thelymphatics that usually occur in newborns.

Cystic tumors tend to grow into the adjacent tissue. Cystic tumorsusually occur in the cervical and axillary region. They can also occurin the soft tissue of the extremities. The main symptoms are dilated,sometimes reticular, structured lymphatics and lymphocysts surrounded byconnective tissue.

Lymphangiomas are assumed to be caused by improperly connected embryoniclymphatics or their deficiency. The result is impaired local lymphdrainage.

Another use for VCC-1 antisense antagonists is in the prevention oftumor angiogenesis, which involves vascularization of a tumor to enableit to growth and/or metastasize. This process is dependent on the growthof new blood vessels. Examples of neoplasms and related conditions thatinvolve tumor angiogenesis include breast carcinomas, lung carcinomas,gastric carcinomas, esophageal carcinomas, colorectal carcinomas, livercarcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervicalcarcinomas, endometrial carcinoma, endometrial hyperplasia,endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendrogliorna, medulloblastoma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors), and Meigs' syndrome.

Healing of trauma such as wound healing and tissue repair is also atargeted use for VCC-1 antisense oligonucleotides. Formation andregression of new blood vessels is essential for tissue healing andrepair. This category includes bone, cartilage, tendon, ligament, and/ornerve tissue growth or regeneration, as well as wound healing and tissuerepair and replacement, and in the treatment of burns, incisions, andulcers.

VCC-1 antisense oligonucleotides that induce cartilage and/or bonegrowth in circumstances where bone is not normally formed haveapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing VCC-1antisense oligonucleotides may have prophylactic use in closed as wellas open fracture reduction and also in the improved fixation ofartificial joints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologic,resection-induced craniofacial defects, and also is useful in cosmeticplastic surgery.

It is expected that VCC-1 antisense oligonucleotides may also exhibitactivity for generation or regeneration of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin, orendothelium), muscle (smooth, skeletal, or cardiac), and vascular(including vascular endothelium) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be byinhibition or modulation of fibrotic scarring to allow normal tissue toregenerate.

VCC-1 antisense oligonucleotides may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage. Also, VCC-1 antisense oligonucleotides may be usefulfor promoting or inhibiting differentiation of tissues described abovefrom precursor tissues or cells, or for inhibiting the growth of tissuesdescribed above.

VCC-1 antisense oligonucleotides may also be used in the treatment ofperiodontal diseases and in other tooth-repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells, or induce differentiation of progenitorsof bone-forming cells VCC-1 antisense oligonucleotides may also beuseful in the treatment of osteoporosis or osteoarthritis, such asthrough stimulation of bone and/or cartilage repair or by blockinginflammation or processes of tissue destruction (collagenase activity,osteoclast activity, etc.) mediated by inflammatory processes, sinceblood vessels play an important role in the regulation of bone turnoverand growth.

Another category of tissue regeneration activity that may beattributable to VCC-1 antisense oligonucleotides is tendon/ligamentformation. A protein that induces tendon/ligament-like tissue or othertissue formation in circumstances where such tissue is not normallyformed has application in the healing of tendon or ligament tears,deformities, and other tendon or ligament defects in humans and otheranimals. Such a preparation may have prophylactic use in preventingdamage to tendon or ligament tissue, as well as use in the improvedfixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of VCC-1antisense oligonucleotides contributes to the repair of congenital,trauma-induced, or other tendon or ligament defects of other origin, andis also useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions herein may provide an environmentto attract tendon- or ligament-forming cells, stimulate growth oftendon- or ligament-forming cells, induce differentiation of progenitorsof tendon- or ligament forming cells, or induce growth oftendon/ligament cells or progenitors ex vivo for return in vivo toeffect tissue repair. The compositions herein may also be useful in thetreatment of tendinitis, carpal tunnel syndrome, and other tendon orligament defects. The compositions may also include an appropriatematrix and/or sequestering agent as a carrier as is well known in theart.

VCC-1 antisense oligonucleotides may also be administeredprophylactically to patients with cardiac hypertrophy, to prevent theprogression of the condition, and avoid sudden death, including death ofasymptomatic patients. Such preventative therapy is particularlywarranted in the case of patients diagnosed with massive leftventricular cardiac hypertrophy (a maximal wall thickness of 35 mm. ormore in adults, or a comparable value in children), or in instances whenthe hemodynamic burden on the heart is particularly strong.

VCC-1 antisense oligonucleotides may also be useful in the management ofatrial fibrillation, which develops in a substantial portion of patientsdiagnosed with hypertrophic cardiomyopathy. Further indications includeangina, myocardial infarctions such as acute myocardial infarctions, andheart failure such as congestive heart failure. Additionalnon-neoplastic conditions include psoriasis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,chronic inflammation, lung inflammation, nephrotic syndrome,preeclampsia, ascites, pericardial effusion (such as that associatedwith pericarditis), and pleural effusion.

In view of the above, VCC-1 antisense oligonucleotides, which are shownto alter or impact endothelial cell function, proliferation, and/orform, are likely to play an important role in the etiology andpathogenesis of many or all of the disorders noted above, and as suchcan serve as therapeutic targets to augment or inhibit these processesor for vascular-related drug targeting in these disorders.

Combination Therapies

The effectiveness of VCC-1 antisense oligonucleotides in preventing ortreating the disorder in question may be improved by administering theactive agent serially or in combination with another agent that iseffective for those purposes, either in the same composition or asseparate compositions. For example, for treatment of cardiachypertrophy, VCC-1 antisense therapy can be combined with theadministration of inhibitors of known cardiac myocyte hypertrophyfactors, e.g., inhibitors of cc-adrenergic agonists such asphenylephrine; endothelin-1 inhibitors such as BOSENTAN™ and MOXONODIN™;inhibitors to CT-I (U.S. Pat. No. 5,679,545); inhibitors to LIF; ACEinhibitors; des-aspartate-angiotensin I inhibitors (U.S. Pat. No.5,773,415), and angiotensin II inhibitors.

For treatment of cardiac hypertrophy associated with hypertension, VCC-1antisense oligonucleotides can be administered in combination withP-adrenergic receptor blocking agents, e.g., propranolol, timolol,tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol,atenolol, metoprolol, or carvedilol; ACE inhibitors, e.g., quinapril,captopril, enalapril, ramipril, benazepril, fosinopril, or lisinopril;diuretics, e.g., chlorothiazide, hydrochlorothiazide,hydroflumethiazide, methylchlothiazide, benzthiazide, dichlorphenamide,acetazolamide, or indapamide; and/or calcium channel blockers, e.g.,diltiazem, nifedipine, verapamil, or nicardipine. Pharmaceuticalcompositions comprising the therapeutic agents identified herein bytheir generic names are commercially available, and are to beadministered following the manufacturers' instructions for dosage,administration, adverse effects, contraindications, etc. 119 See, e.z.,Physicians' Desk Reference (Medical Economics Data Production Co.:Montvale, N.J., 1997), 51 st Edition. Preferred candidates forcombination therapy in the treatment of hypertrophic cardiomyopathy areP-adrenergic-blocking drugs (e.g., propranolol, timolol, tertalolol,carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol,metoprolol, or carvedilol), verapamil, difedipine, or diltiazem.Treatment of hypertrophy associated with high blood pressure may requirethe use of antihypertensive drug therapy, using calcium channelblockers, e.g., diltiazem, nifedipine, verapamil, or nicardipine;P-adrenergic blocking agents; diuretics, e.g., chlorothiazide,hydrochlorothiazide, hydroflumethiazide, methylchlothiazide,benzthiazide, dichlorphenamide, acetazolamide, or indapamide; and/orACE-inhibitors, e. g., quinapril, captopril, enalapril, ramipril,benazepril, fosinopril, or lisinopril.

For other indications, VCC-1 antisense oligonucleotides may be combinedwith other agents beneficial to the treatment of the bone and/orcartilage defect, wound, or tissue in question. These agents includevarious growth factors such as EGF, PDGF, TGF- or TGF-, IGF, FGF, andCTGF.

In addition, VCC-1 antisense oligonucleotides used to treat cancer maybe combined with cytotoxic, chemotherapeutic, or growth-inhibitoryagents as identified above. Also, for cancer treatment, VCC-1 antisenseoligonucleotides are suitably administered serially or in combinationwith radiological treatments, whether involving irradiation oradministration of radioactive substances.

The effective amounts of the therapeutic agents administered incombination with VCC-1 antisense oligonucleotides thereof will be at thephysician's, or veterinarian's discretion. Dosage administration andadjustment is done to achieve maximal management of the conditions to betreated. For example, for treating hypertension, these amounts ideallytake into account use of diuretics or digitalis, and conditions such ashyper- or hypotension, renal impairment, etc. The dose will additionallydepend on such factors as the type of the therapeutic agent to be usedand the specific patient being treated. Typically, the amount employedwill be the same dose as that used, if the given therapeutic agent isadministered without VCC-1 antisense oligonucleotides.

For treatment of breast carcinoma, VCC-1 antisense oligonucleotides canbe administered in combination with, but not limited to, Trastuzumab(Herceptin) with chemotherapy, paclitaxel, docetaxel, epirubicin,mitoxantrone, topotecan, capecitabine, vinorelbine, thiotepa,vincristine, vinblastine, carboplatin or cisplatin, plicamycin,anastrozole, letrozole, exemestane, toremifine, or progestins.

For treatment of acute lymphocytic leukemia, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, doxorubicin, cytarabine, cyclophosphamide, etoposide,teniposide, allopurinol, or autologous bone marrow transplantation.

For treatment of acute myelocytic and myelomonocytic leukemia, VCC-1,antisense oligonucleotides can be administered in combination with, butnot limited to, gemtuzumab ozogamicin (Mylotarg), mitoxantrone,idarubicin, etoposide, mercaptopurine, thioguanine, azacitidine,amsacrine, methotrexate, doxorubicin, tretinoin, allopurinol,leukapheresis, prednisone, or arsenic trioxide for acute promyelocyticleukemia.

For treatment of chronic myelocytic leukemia, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, busulfan, mercaptopurine, thioguanine, cytarabine,plicamycin, melphalan, autologous bone marrow transplantation, orallopurinol.

For treatment of chronic lymphocytic leukemia, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, vincristine, cyclophosphamide, doxorubicin, cladribine(2-chlorodeoxyadenosine; CdA), allogeneic bone marrow transplant,androgens, or allopurinol.

For treatment of multiple myeloma, VCC-1 antisense oligonucleotides canbe administered in combination with, but not limited to, etoposide,cytarabine, alpha interferon, dexamethasone, or autologous bone marrowtransplantation.

For treatment of carcinoma of the lung (small cell and non-small cell),VCC-1 antisense oligonucleotides can be administered in combinationwith, but not limited to, cyclophosphamide, doxorubicin, vincristine,etoposide, mitomycin, ifosfamide, paclitaxel, irinotecan, or radiationtherapy.

For treatment of carcinoma of the colon and rectum, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, capecitabine, methotrexate, mitomycin, carmustine,cisplatin, irinotecan, or floxuridine.

For treatment of carcinoma of the kidney, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, alpha interferon, progestins, infusional FUDR, orfluorouracil.

For treatment of carcinoma of the prostate, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, ketoconazole, doxorubicin, aminoglutethimide, progestins,cyclophosphamide, cisplatin, vinblastine, etoposide, suramin, PC-SPES,or estramustine phosphate.

For treatment of melanoma, VCC-1 antisense oligonucleotides can beadministered in combination with, but not limited to, carmustine,lomustine, melphalan, thiotepa, cisplatin, paclitaxel, tamoxifen, orvincristine.

For treatment of carcinoma of the ovary, VCC-1 antisenseoligonucleotides can be administered in combination with, but notlimited to, docetaxel, doxorubicin, topotecan, cyclophosphamide,doxorubicin, etoposide, or liposomal doxorubicin.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases (i.e.from about 8 to about 30 linked nucleo sides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 25 nucleobases. As is known inthe art, a nucleoside is a base-sugar combination. The base portion ofthe nucleoside is normally a heterocyclic base. The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure, however, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside backboneof the oligonucleotide. The normal I linkage or backbone of RNA and DNAis a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n),OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH_(2n)ON[(CH₂)_(n)CH₃)]₂ where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀, (lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ON0₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharnacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below,and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂-O—CH₂-N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy(2′-O CH₃),2′-aminopropoxy(2′-O CH₂ CH₂ CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of whichis herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 365'-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,365'-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds, which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease, which cleaves the RNAstrand of RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently, and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 119). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutanic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, and prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder, which can be treated by modulating theexpression of VCC-1, is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingVCC-1, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding VCC-1 can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of VCC-1 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations, which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutions,which may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances, which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention. Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug, which may be present as a solution in either theaqueous phase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosaqe Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, non-swelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifing materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives, andantioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral, andparenteral routes and methods for their manufacture has been reviewed inthe literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 199). Emulsion formulations for oral delivery have been very widelyused because of reasons of ease of formulation, efficacy from anabsorption and bioavailability standpoint. (Rosoff, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,oil-soluble vitamins and high fat nutritive preparations are among thematerials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphile,which is a single optically isotropic, and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 1852-5).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtriglycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Noncationic liposomes, although not able to fuse as efficiently with thecell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome, which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, P. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action: Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes, which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985)

Liposomes, which are pH-sensitive or negatively charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) was ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term, which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such, specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., =i Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside Gjor a galactocerebroside sulfateester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂ 15G thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid droplets,which are so highly deformable that they are easily able to penetratethrough pores that are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285)

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285). Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids particularlyoligonucleotides, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating nonsurfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., JPharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-.rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcamitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiologicalrole of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds. McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate' and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Canier Systems, 1991, page 92;Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashitaet al., J Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium. ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9, and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, nonchelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers includes, for example, unsaturated cyclicureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin, and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′sothiocyano-stilbene-2,2′disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration, which does not deleteriously react withnucleic acids, can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration, which do not deleteriously react with nucleic acids, canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention.' The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances, which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol, and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites areavailable from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotidesis utilized, except the wait step after pulse delivery of tetrazole andbase is increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me—C)nucleotides are synthesized according to published methods [Sanghvi, et.al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides are synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine is synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenineis selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups is accomplished using standard methodologies andstandard methods are used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine is accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group is followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation is followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies are used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by themodification of a literature procedure in which2,2′anhydro-1-beta-D-arabinofuranosyluracil is treated with 70% hydrogenfluoride-pyridine. Standard procedures are used to obtain the 5′-DMT and5′-DMT-3′-phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine is synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures are used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridinel

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) are added to DMF (300 mL). Themixture is heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution is concentrated under reduced pressure. Theresulting syrup is poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether is decanted and the residue is dissolvedin a minimum amount of methanol (ca. 400 mL). The solution is pouredinto fresh ether (2.5 L) to yield a stiff gum. The ether is decanted andthe gum is dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give asolid that is crushed to a light tan powder. The material is used as isfor further reactions (or it can be purified further by columnchromatography using a gradient of methanol in ethyl acetate (10-25%) togive a white solid.

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) are added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel is openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue is suspended in hot acetone (1 L). The insoluble saltsare filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) is dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) is packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue is dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct is eluted with the packing solvent to give the title product.Additional material can be obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) is co-evaporated withpyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). Afirst aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) is added andthe mixture stirred at room temperature for one hour. A second aliquotof dimethoxytrityl chloride (94.3 g, 0.278 M) is added and the reactionstirred for an additional one hour. Methanol (170 mL) is then added tostop the reaction. The solvent is evaporated and triturated with CH₃CN(200 mL) The residue is dissolved in CHCl (1.5 L) and extracted with2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organicphase is dried over Na₂SO₄, filtered, and evaporated. The residue ispurified on a 3.5 kg silica gel column, packed and eluted withEtOAc/hexane/acetone (5:5:1) containing 0-5% Et₃NH. The pure fractionsare evaporated to give the title product.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) arecombined and stirred at room temperature for 24 hours. The reaction ismonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) isadded and the mixture evaporated at 35° C. The residue is dissolved inCHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers are backextracted with 200 mL of CHCl₃. The combined organics are dried withsodium sulfate and evaporated to a residue. The residue is purified on a3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pureproduct fractions are evaporated to yield the title compounds.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyltriazoleuridine

A first solution is prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) is added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃is added dropwise, over a 30 minute period, to the stirred solutionmaintained at O-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution is added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixture isstored overnight in a cold room. Salts are filtered from the reactionmixture and the solution is evaporated. The residue is dissolved inEtOAc (1 L) and the insoluble solids are removed by filtration. Thefiltrate is washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue istriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH40H (30 mL) is stirred atroom temperature for 2 hours. The dioxane solution is evaporated and theresidue azeotroped with MeOH (2×200 mL). The residue is dissolved inMeOH (300 mL) and transferred to a 2-liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas is added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents are evaporated to dryness and the residue is dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics are dried over sodium sulfate and the solvent is evaporated togive the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)is dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) isadded with stirring. After stirring for 3 hours, TLC showed the reactionto be approximately 95% complete. The solvent is evaporated and theresidue azeotroped with MeOH (200 mL). The residue is dissolved in CHCl₃(700 mL) and extracted with saturated NaHCO, (2×300 mL) and saturatedNaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue. Theresidue is chromatographed on a 1.5 kg silica column using EtOAc/hexane(1:1) containing 0-5% Et₃NH as the eluting solvent. The pure productfractions are evaporated to give the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) is dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) areadded with stirring, under a nitrogen atmosphere. The resulting mixtureis stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture is extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes areback-extracted with CH₂Cl₂ (300 mL), and the extracts are combined,dried over MgSO₄ and concentrated. The residue obtained ischromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give the titlecompound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites2′-(D)imethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.4'6 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) aredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) is added in one portion. Thereaction is stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethylacetate) indicated a complete reaction. The solution is concentratedunder reduced pressure to a thick oil. This is partitioned betweendichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine(1 L). The organic layer is dried over sodium sulfate and concentratedunder reduced pressure to a thick oil. The oil is dissolved in a 1:1mixture of ethyl acetate and ethyl ether (600 mL) and the solution iscooled to −10° C. The resulting crystalline product is collected byfiltration, washed with ethyl ether (3×200 mL), and dried (40° C., 1 mmHg, 24 h) to a white solid

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor is added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) is added cautiously atfirst until the evolution of hydrogen gas subsides.5′-O-tert-Butyldiphenylsilyl-O²-2′anhydro-5-methyluridine (149 g, 0.3'1mol) and sodium bicarbonate (0.074 g, 0.003 eq) are added with manualstirring. The reactor is sealed and heated in an oil bath until aninternal temperature of 160° C. is reached and then maintained for 16 h(pressure <100 psig). The reaction vessel is cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction isstopped, concentrated under reduced pressure (10 to 1 mm, Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue ispurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions arecombined, stripped and dried to product as a white crisp foam,contaminated starting material, and pure reusable starting material.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) is mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It is then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture isflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) isadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) is added dropwise to the reaction mixture. The rate of addition ismaintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition is complete, thereaction is stirred for 4 hrs. By that time TLC showed the completion ofthe reaction (ethylacetate:hexane, 60:40). The solvent is evaporated invacuum. Residue obtained is placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam.

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) is dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) is added dropwise at −10° C. to 0°C. After 1 h the mixture is filtered, the filtrate is washed with icecold CH₂Cl₂ and the combined organic phase is washed with water, brineand dried over anhydrous Na₂SO₄. The solution is concentrated to get2′-O(aminooxyethyl)thymidine, which is then dissolved in MeOH (67.5 mL).To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) is added andthe resulting mixture is stirred for 1 h. Solvent is removed undervacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam.

5′-O-tert-Butyldiphenyisilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) is dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) is added to this solution at 10° C. under inertatmosphere. The reaction mixture is stirred for 10 minutes at 10° C.After that the reaction vessel is removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) is added and extracted withethyl acetate (2×20 mL). Ethyl acetate phase is dried over anhydrousNa₂SO₄, evaporated to dryness. Residue is dissolved in a solution of 1MPPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) isadded and the reaction mixture is stirred at room temperature for 10minutes. Reaction mixture cooled to 10° C. in an ice bath, sodiumcyanoborohydride (0.39 g, 6.13 mmol) is added, and reaction mixturestirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixtureis removed from the ice bath and stirred at room temperature for 2 hrs.To the reaction mixture 5% NaHCO₃ (25 mL) solution is added andextracted with ethyl acetate (2×25 mL). Ethyl acetate layer is driedover anhydrous Na₂SO₄ and evaporated to dryness. The residue obtained ispurified by flash column chromatography and eluted with 5% MeOH inCH₂Cl₂ to get5′-O-tertbutyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam.

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) is dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF is then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionis monitored by TLC (5% MeOH in CH₂Cl₂). Solvent is removed under vacuumand the residue placed on a flash column and eluted with 10% MeOH inCH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine.

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) isdried over P₂O₅ under high vacuum overnight at 40° C. It is thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained isdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) is added to the mixture and the reactionmixture is stirred at room temperature until all of the startingmaterial disappeared. Pyridine is removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-0(dimethylamino-oxyethyl)-5-methyluridine.

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) is co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) is added and driedover P20, under high vacuum overnight at 40° C. Then the reactionmixture is dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) is added. The reaction mixture is stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction ismonitored by TLC (hexane:ethyl acetate 1:1). The solvent is evaporated,then the residue is dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer is dried over anhydrousNa₂SO₄ and concentrated. Residue obtained is chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite)as a foam.

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine ribosidemay be resolved and converted to 2′-O-(2ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramiditel.

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaninoethoxyethyl, i.e., 2′O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxylethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath, and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)1-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH: CH₂Cl₂:Et₃N (20:1, v/v, with 1%triethylamine) gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxyN,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle is replaced by 0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe stepwise thiation of the phosphite linkages. The thiation wait stepis increased to 68 sec and is followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides are purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described in WO94/17093 and WO 94/02499 herein incorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825; 5,386,023; 5,489,677; 5,602,240; and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 523. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082; 5,700,922; and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample is again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides are prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of phorothioate oligonucleotides are prepared as per theprocedure above for 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)] Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides are analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85%full-length material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis are periodically checkedby “P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides are purified by HPLC, as described by Chiang et al., J.Biol. Chem. 1991, 266, 18162-18171.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides are synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages are afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages are generated by sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) inanhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites can be purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected betacyanoethyldiisopropyl phosphoramidites.

Oligonucleotides are cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well is assessed bydilution of samples and LW absorption spectroscopy. The full-lengthintegrity of the individual products is evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates are diluted from the master plateusing single and multi-channel robotic pipettors. Plates are judged tobe acceptable if at least 85% of the compounds on the plate are at least85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing 6 cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, Ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 is obtainedfrom the American Type Culture Collection (ATCC) (Manassas, Va.). T-24cells are routinely cultured in complete McCoy's 5A basal media(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetalcalf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100units per mL, and streptomycin 100 micrograms per mL (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells are routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells areseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 can be obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells areroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells are routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) can be obtained from theClonetics Corporation (Walkersville Md.). NHDFs are routinely maintainedin Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells are maintained for upto 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) can be obtained from the CloneticsCorporation (Walkersville Md.). HEKs are routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells are routinelymaintained for up to 10 passages as recommended by the supplier.

MCF-7 Cells:

The human breast carcinoma cell line MCF-7 is obtained from the AmericanType Culture Collection (Manassas, Va.). MCF-7 cells are routinelycultured in DMEM low glucose (Gibco/Life Technologies, Gaithersburg,Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies,Gaithersburg, Md.). Cells are routinely passaged by typsinization anddilution when they reached 90% confluence. Cells are seeded into 96-wellplates (Falcon-Primaria #3872) at a density of 7000 cells/well for usein RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

LA4 Cells:

The mouse lung epithelial cell line LA4 is obtained from the 20 AmericanType Culture Collection (Manassas, Va.). LA4 cells are routinelycultured in F 12K medium (Gibco/Life Technologies, Gaithersburg, Md.)supplemented with 15% fetal calf serum (Gibco/Life Technologies,Gaithersburg, Md.). Cells are routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells are seeded into 96-wellplates (Falcon-Primaria #3872) at a density of 3000-6000 cells/ well foruse in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 80% confluence, they are treated witholigonucleotide. For cells grown in 96-well plates, wells are washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL, LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium is replaced with fresh medium. Cells areharvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations.

Example 10 Analysis of Oligonucleotide Inhibition of VCC-1 Expression

Antisense modulation of VCC-1 expression can be assayed in a variety ofways known in the art. For example, VCC-1 mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions. Prior to quantitative PCR analysis, primer-probe setsspecific to the target gene being measured are evaluated for theirability to be “multiplexed” with a GAPDH amplification reaction. Inmultiplexing, both the target gene and the internal standard gene GAPDHare amplified concurrently in a single sample. In this analysis, mRNAisolated from untreated cells is serially diluted. Each dilution isamplified in the presence of primer-probe sets specific for GAPDH only,target gene only (“single-plexing”), or both (multiplexing). FollowingPCR amplification, standard curves of GAPDH and target mRNA signal as afunction of dilution are generated from both the single-plexed andmultiplexed samples. If both the slope and correlation coefficient ofthe GAPDH and target signals generated from the multiplexed samples fallwithin 10% of their corresponding values generated from thesingle-plexed samples, the primer-probe set specific for that target isdeemed as multiplexable. Other methods of PCR are also known in the art.

Protein levels of VCC-1 can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to VCC-1 can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.110.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley Sons, Inc.,1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+mRNA Isolation

Poly(A)+mRNA is isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium is removedfrom the cells and each well is washed with 200 μL cold PBS. 60 μL lysisbuffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mMvanadyl-ribonucleoside complex) is added to each well, the plate isgently agitated and then incubated at room temperature for five minutes.55 μL of lysate is transferred to Oligo d(T) coated 96-well plates (AGCTInc., Irvine Calif.). Plates are incubated for 60 minutes at roomtemperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HClpH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate isblotted on paper towels to remove excess wash buffer and then air-driedfor 5 minutes. 60 pL of elution buffer (5 mM Tris-HCl pH 7.6), preheatedto 70° C. is added to each well, the plate is incubated on a 90° C. hotplate for 5 minutes, and the eluate is then transferred to a fresh96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total mRNA is isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium is removed from the cells and each well is washed with 200μL cold PBS. 100 μL Buffer RLT is added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol is then addedto each well and the contents mixed by pipetting three times up anddown. The samples are then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum is applied for 15 seconds. 1 mL ofBuffer RW1 is added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 nL of Buffer RPE is then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash is then repeated and the vacuum is appliedfor an additional 10 minutes. The plate is then removed from the QIAVAC™manifold and blotted dry on paper towels. The plate is then re-attachedto the QIAVAC™ manifold fitted with a collection tube rack containing1.2 mL collection tubes. RNA is then eluted by pipetting 60 μL waterinto each well, incubating 1 minute, and then applying the vacuum for 30seconds. The elution step is repeated with an additional 60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of VCC-1 mRNA Levels

Quantitation of VCC-1 mRNA levels is determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM™, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

PCR reagents can be obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions are carried out by adding 25 μL PCR cocktail (1×TAQMAN™ buffer A, 5.5 MM MgCl₂, 300 μM each of DATP, dCTP and dGTP, 600μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLVreverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNAsolution. The RT reaction is carried out by incubation for 30 minutes at48° C. Following a 10 minute incubation at 95° C. to activate theAMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol are carried out:95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes(annealing/extension).

Probes and primers to human VCC-1 were designed to hybridize to a humanVCC-1 sequence, using published sequence, information (GenBank accessionnumber XM_(—)058945, incorporated herein as FIG. 1. For human VCC-1 thePCR primers were: SEQ ID NO: 1100 forward primer: CGACAGTTGCGATGAAAGTTCTSEQ ID NO: 1101 reverse primer: AGAGACCATGGACATCAGCATTAG and SEQ ID NO:1102 the PCR probe is: FAM ™-TCTCTTCCCTCCTCCTGTTGCTGCC-TAMRA

and the PCR probe is: FAM™-TCTCTTCCCTCCTCCTGTTGCTGCC SEQ ID NO:1102-TAMRA where FAM™ (PE-Applied Biosystems, Foster City, Calif.) isthe fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, FosterCity, Calif.) is the quencher dye. For human cyclophilin the PCR primerswere: +TR,1SEQ ID NO:1103 forward primer: CCCACCGTGTTCTTCGACAT +TR,1SEQID NO:1104 reverse primer: TTTCTGCTGTCTTTGGGACCTT and +TR,1SEQ IDNO:1105 the PCR probe is: 5′ JOE-CGCGTCTCCTTTGAGCTGTTTGCA-TAMRA 3′the PCR probe is: 5′ JOE-CGCGTCTCCTTTGAGCTGTTTGCA SEQ ID NO: 1105-TAMRA3′ where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14 Antisense Inhibition of Human VCC-1 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotidesare designed to target different regions of the human VCC-1 RNA, usingpublished sequences (XM_(—)058945, incorporated herein as FIG. 1. Theoligonucleotides are shown in Table 1. “Position” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. The indicated parameters for each oligo werepredicted using RNAstructure 3.7 by David H. Mathews, Michael Zuker, andDouglas H. Turner. The parameters are described either as free energy(The energy that is released when a reaction occurs. The more negativethe number, the more likely the reaction will occur. All free energyunits are in kcal/mol.) or melting temperature (The temperature at whichtwo anneal strands of polynucleic acid separate. The higher thetemperature, greater the affinity between the 2 strands.) When designingan antisense oligonucleotide that will bind with high affinity, it isdesirable to consider the structure of the target RNA strand and theantisense oligomer. Specifically, for an oligomer to bind tightly (inthe table described as ‘duplex formation’), it should be complementaryto a stretch of target RNA that has little self-structure (in the tablethe free energy of which is described as ‘target structure’). Also, theoligomer should have little self-structure, either intramolecular (inthe table the free energy of which is described as ‘intramolecularoligo’) or bimolecular (in the table the free energy of which isdescribed as ‘intermolecular oligo’). Breaking up any self-structureamounts to a binding penalty. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. Cytidine residues in the 2′-MOE wingsare 5-methylcytidines. All cytidine residues are 5-methylcytidines.TABLE 1 duplex target Intra- Inter- total forma- Tm of struc- molecularmolecular position oligo binding tion Duplex ture oligo oligo 414CTGTGGTGCCTTTGGTGTCT −26.2 −28.3 82.5 −2.1 0 −5.7 SEQ ID NO:1 419GCTTTCTGTGGTGCCTTTGG −25.8 −27.9 80.7 −2.1 0 −5.7 SEQ ID NO:2 415TCTGTGGTGCCTTTGGTGTC −25.7 −27.8 82.4 −2.1 0 −5 SEQ ID NO:3 410GGTGCCTTTGGTGTCTTGTT −25.5 −27.6 81.5 −2.1 0 −4.9 SEQ ID NO:4 411TGGTGCCTTTGGTGTCTTGT −25.4 −27.5 80.8 −2.1 0 −5.7 SEQ ID NO:5 412GTGGTGCCTTTGGTGTCTTG −25.4 −27.5 80.8 −2.1 0 −5.7 SEQ ID NO:6 413TGTGGTGCCTTTGGTGTCTT −25.4 −27.5 80.8 −2.1 0 −5.7 SEQ ID NO:7 416TTCTGTGGTGCCTTTGGTGT −25.4 −27.5 80.8 −2.1 0 −5.7 SEQ ID NO:8 418CTTTCTGTGGTGCCTTTGGT −25.2 −27.3 79.8 −2.1 0 −5.7 SEQ ID NO:9 424GTTTGGCTTTCTGTGGTGCC −24.8 −28.2 82.4 −2.1 −1.2 −5.2 SEQ ID NO:10 956GTGAGGGTCTTGGTGGGGAT −24.7 −27.4 80.4 −2.7 0 −2.4 SEQ ID NO:11 409GTGCCTTTGGTGTCTTGTTT −24.4 −26.5 79.1 −2.1 0 −3.4 SEQ ID NO:12 420GGCTTTCTGTGGTGCCTTTG −24.4 −27.9 80.7 −2.1 −1.3 −5.7 SEQ ID NO:13 417TTTCTGTGGTGCCTTTGGTG −24.3 −26.4 77.5 −2.1 0 −5.7 SEQ ID NO:14 425TGTTTGGCTTTCTGTGGTGC −24.1 −26.2 78.4 −2.1 0 −3.7 SEQ ID NO:15 421TGGCTTTCTGTGGTGCCTTT −23.8 −27.9 80.7 −2.1 −2 −6 SEQ ID NO:16 422TTGGCTTTCTGTGGTGCCTT −23.8 −27.9 80.7 −2.1 −2 −6 SEQ ID NO:17 423TTTGGCTTTCTGTGGTGCCT −23.8 −27.9 80.7 −2.1 −2 −6 SEQ ID NO:18 407GCCTTTGGTGTCTTGTTTTC −23.7 −25.8 77.8 −2.1 0 −3.2 SEQ ID NO:19 957AGTGAGGGTCTTGGTGGGGA −23.4 −27.4 80.8 −4 0 −2.4 SEQ ID NO:20 408TGCCTTTGGTGTCTTGTTTT −23.3 −25.4 75.7 −2.1 0 −3.4 SEQ ID NO:21 955TGAGGGTCTTGGTGGGGATA −23.2 −25.9 76 −2.7 0 −2.4 SEQ ID NO:22 952GGGTCTTGGTGGGGATAAGT −23.1 −25.8 75.8 −2.7 0 −3.2 SEQ ID NO:23 171GGCAGCAACAGGAGGAGGGA −22.6 −27 75.9 −4.4 0 −5.3 SEQ ID NO:24 566GAGTGTCTGGTAGGTGTGCT −22.5 −26.7 81.5 −4.2 0 −3.6 SEQ ID NO:25 954GAGGGTCTTGGTGGGGATAA −22.5 −25.2 73.6 −2.7 0 −2.4 SEQ ID NO:26 426TTGTTTGGCTTTCTGTGGTG −22.4 −24.5 74 −2.1 0 −3.7 SEQ ID NO:27 565AGTGTCTGGTAGGTGTGCTC −22.3 −26.5 82.1 −4.2 0 −3.6 SEQ ID NO:28 403TTGGTGTCTTGTTTTCTTCA −22.2 −23.1 72 −0.7 0 −1.9 SEQ ID NO:29 404TTTGGTGTCTTGTTTTCTTC −22.1 −22.5 71.2 0 0 −1.5 SEQ ID NO:30 613GAATGATTTAGGGGTGGGTA −22.1 −22.5 67 0 0 −2.1 SEQ ID NO:31 172TGGCAGCAACAGGAGGAGGG −22 −26.4 74.4 −4.4 0 −5.3 SEQ ID NO:32 614GGAATGATTTAGGGGTGGGT −22 −24 70.2 −2 0 −2.3 SEQ ID NO:33 889GGGTCATCTGGTTGTGAATT −21.9 −23.7 71 −1.8 0 −3.3 SEQ ID NO:34 953AGGGTCTTGGTGGGGATAAG −21.9 −24.6 72.5 −2.7 0 −2.4 SEQ ID NO:35 1CGTTCCCATTTGAGGGCGAG −21.8 −27.6 74.4 −4.5 −1.2 −6.4 SEQ ID NO:36 890TGGGTCATCTGGTTGTGAAT −21.8 −23.6 70.4 −1.8 0 −3.3 SEQ ID NO:37 891ATGGGTCATCTGGTTGTGAA −21.8 −23.6 70.4 −1.8 0 −3.3 SEQ ID NO:38 892AATGGGTCATCTGGTTGTGA −21.8 −23.6 70.4 −1.8 0 −3.3 SEQ ID NO:39 567AGAGTGTCTGGTAGGTGTGC −21.6 −25.8 79.6 −4.2 0 −2.6 SEQ ID NO:40 951GGTCTTGGTGGGGATAAGTA −21.6 −24.3 72.4 −2.7 0 −3.2 SEQ ID NO:41 715CTGGGTAAGGGGAGGGCACA −21.5 −27.5 77 −6 0 −4 SEQ ID NO:42 958GAGTGAGGGTCTTGGTGGGG −21.4 −27.4 80.8 −6 0 −2.2 SEQ ID NO:43 405CTTTGGTGTCTTGTTTTCTT −21.3 −23 71.5 −1.7 0 −1.3 SEQ ID NO:44 174AGTGGCAGCAACAGGAGGAG −21 −25.2 72.9 −4.2 0 −2.4 SEQ ID NO:45 562GTCTGGTAGGTGTGCTCACT −20.9 −27.1 81.9 −4.2 −2 −4.2 SEQ ID NO:46 173GTGGCAGCAACAGGAGGAGG −20.8 −26.4 75.3 −5.6 0 −6.1 SEQ ID NO:47 161GGAGGAGGGAAGAGATTAGA −20.7 −21.5 64.7 −0.6 0 −1.5 SEQ ID NO:48 170GCAGCAACAGGAGGAGGGAA −20.7 −25.1 71 −4.4 0 −4.7 SEQ ID NO:49 175TAGTGGCAGCAACAGGAGGA −20.7 −24.9 72 −4.2 0 −2.4 SEQ ID NO:50 888GGTCATCTGGTTGTGAATTG −20.7 −22.5 68.1 −1.8 0 −3.1 SEQ ID NO:51 714TGGGTAAGGGGAGGGCACAG −20.6 −26.6 75.4 −6 0 −4 SEQ ID NO:52 897GGTAAAATGGGTCATCTGGT −20.6 −22.4 66.3 −1.8 0 −2.9 SEQ ID NO:53 898GGGTAAAATGGGTCATCTGG −20.6 −22.4 65.7 −1.8 0 −2.9 SEQ ID NO:54 227GGCCTCTGGCGACCCCTGGA −20.5 −34.5 87.6 −11.5 −2.5 −8.4 SEQ ID NO:55 564GTGTCTGGTAGGTGTGCTCA −20.5 −27.2 82.9 −6.7 0 −0.6 SEQ ID NO:56 893AAATGGGTCATCTGGTTGTG −20.5 −22.3 66.7 −1.8 0 −2.9 SEQ ID NO:57 950GTCTTGGTGGGGATAAGTAT −20.4 −23.1 69.6 −2.7 0 −3.2 SEQ ID NO:58 946TGGTGGGGATAAGTATGTGT −20.2 −22.9 68.7 −2.7 0 −1.8 SEQ ID NO:59 162AGGAGGAGGGAAGAGATTAG −20.1 −20.9 63.6 −0.6 0 −1.5 SEQ ID NO:60 226GCCTCTGGCGACCCCTGGAT −20.1 −33.3 85.2 −11.5 −1.7 −7.8 SEQ ID NO:61 612AATGATTTAGGGGTGGGTAC −20.1 −22.1 66.2 −2 0 −4 SEQ ID NO:62 948CTTGGTGGGGATAAGTATGT −20 −22.7 67.8 −2.7 0 −2.1 SEQ ID NO:63 228TGGCCTCTGGCGACCCCTGG −19.9 −33.9 86.2 −11.5 −2.5 −8.1 SEQ ID NO:64 229GTGGCCTCTGGCGACCCCTG −19.9 −33.9 87.2 −11.5 −2.5 −8.3 SEQ ID NO:65 402TGGTGTCTTGTTTTCTTCAC −19.9 −23.2 72.3 −3.3 0 −3.6 SEQ ID NO:66 427CTTGTTTGGCTTTCTGTGGT −19.9 −25.4 76.3 −5.5 0 −3.7 SEQ ID NO:67 560CTGGTAGGTGTGCTCACTGT −19.9 −26.7 79.6 −4.8 −2 −4.2 SEQ ID NO:68 945GGTGGGGATAAGTATGTGTA −19.9 −22.6 68.2 −2.7 0 −1.8 SEQ ID NO:69 135ATCGCAACTGTCGGTGCAGC −19.8 −27.2 75.3 −5.8 −1.6 −6.8 SEQ ID NO:70 406CCTTTGGTGTCTTGTTTTCT −19.8 −24.9 75.1 −5.1 0 −2 SEQ ID NO:71 606TTAGGGGTGGGTACAGTGGG −19.8 −26.4 77.4 −5.9 −0.4 −5.2 SEQ ID NO:72 894AAAATGGGTCATCTGGTTGT −19.8 −21.6 64.5 −1.8 0 −2.9 SEQ ID NO:73 2GCGTTCCCATTTGAGGGCGA −19.7 −29.4 78.2 −8.2 −1.4 −7.1 SEQ ID NO:74 401GGTGTCTTGTTTTCTTCACA −19.7 −23.9 73.7 −3 −1.1 −4.7 SEQ ID NO:75 561TCTGGTAGGTGTGCTCACTG −19.7 −25.9 77.7 −4.2 −2 −4.2 SEQ ID NO:76 225CCTCTGGCGACCCCTGGATT −19.6 −31.6 81.5 −11.5 −0.1 −4.5 SEQ ID NO:77 137TCATCGCAACTGTCGGTGCA −19.5 −26.5 73.5 −5.8 −1.1 −7 SEQ ID NO:78 605TAGGGGTGGGTACAGTGGGA −19.5 −26.9 78.5 −7.4 0.2 −5.2 SEQ ID NO:79 896GTAAAATGGGTCATCTGGTT −19.5 −21.3 64.1 −1.8 0 −2.9 SEQ ID NO:80 1048GTATGCTTTTTTTTTTTTGT −19.5 −19.9 63.1 0 0 −3.6 SEQ ID NO:81 1049GGTATGCTTTTTTTTTTTTG −19.5 −19.9 62.5 0 0 −2.9 SEQ ID NO:82 1050TGGTATGCTTTTTTTTTTTT −19.5 −19.9 62.5 0 0 −3.6 SEQ ID NO:83 1051TTGGTATGCTTTTTTTTTTT −19.5 −19.9 62.5 0 0 −3.6 SEQ ID NO:84 132GCAACTGTCGGTGCAGCTGT −19.4 −28.1 79.1 −7.3 −1.3 −9.7 SEQ ID NO:85 899AGGGTAAAATGGGTCATCTG −19.4 −21.2 63.4 −1.8 0 −2.9 SEQ ID NO:86 140CTTTCATCGCAACTGTCGGT −19.3 −25.1 71 −5.8 0 −4.7 SEQ ID NO:87 158GGAGGGAAGAGATTAGAACT −19.3 −20.1 60.9 −0.6 0 −2.3 SEQ ID NO:88 965GGAGACAGAGTGAGGGTCTT −19.3 −24.7 74.4 −3.9 −1.4 −5.5 SEQ ID NO:89 138TTCATCGCAACTGTCGGTGC −19.2 −25.9 72.8 −5.8 −0.8 −7 SEQ ID NO:90 176TTAGTGGCAGCAACAGGAGG −19.2 −24.4 71 −5.2 0 −2.4 SEQ ID NO:91 949TCTTGGTGGGGATAAGTATG −19.2 −21.9 66.1 −2.7 0 −2.7 SEQ ID NO:92 963AGACAGAGTGAGGGTCTTGG −19.2 −24.1 72.7 −3.9 −0.9 −5.1 SEQ ID NO:93 400GTGTCTTGTTTTCTTCACAT −19.1 −22.7 70.8 −3 −0.3 −3.9 SEQ ID NO:94 611ATGATTTAGGGGTGGGTACA −19.1 −23.5 69.8 −3.7 −0.4 −5.2 SEQ ID NO:95 615TGGAATGATTTAGGGGTGGG −19.1 −22.8 66.8 −3.7 0 −2.3 SEQ ID NO:96 900TAGGGTAAAATGGGTCATCT −19.1 −20.9 62.9 −1.8 0 −2.9 SEQ ID NO:97 947TTGGTGGGGATAAGTATGTG −19.1 −21.8 65.7 −2.7 0 −1.8 SEQ ID NO:98 962GACAGAGTGAGGGTCTTGGT −19 −25.3 76.1 −5.8 −0.1 −4.4 SEQ ID NO:99 169CAGCAACAGGAGGAGGGAAG −18.9 −23.3 67.1 −4.4 0 −4.1 SEQ ID NO:100 160GAGGAGGGAAGAGATTAGAA −18.8 −19.6 60 −0.6 0 −1.5 SEQ ID NO:101 168AGCAACAGGAGGAGGGAAGA −18.8 −23.2 67.2 −4.4 0 −4.1 SEQ ID NO:102 887GTCATCTGGTTGTGAATTGG −18.8 −22.5 68.1 −3.7 0 −3.1 SEQ ID NO:103 1065CCGTGTCTGGTTCATTGGTA −18.8 −26.3 76 −7.5 0 −2.9 SEQ ID NO:104 64TCCCTGGGGATGACTCAGGT −18.7 −28.7 80.3 −6.9 −3.1 −9.3 SEQ ID NO:105 136CATCGCAACTGTCGGTGCAG −18.7 −26.1 72.2 −5.8 −1.6 −8.4 SEQ ID NO:106 607TTTAGGGGTGGGTACAGTGG −18.7 −25.3 75.1 −5.9 −0.4 −5.2 SEQ ID NO:107 1061GTCTGGTTCATTGGTATGCT −18.7 −25 75.5 −5.8 −0.1 −3.6 SEQ ID NO:108 568AAGAGTGTCTGGTAGGTGTG −18.5 −23.3 71.8 −4.8 0 −2.9 SEQ ID NO:109 685GACGAGAGAAGAAGACACTA −18.5 −18.9 57.3 0 0 −3.5 SEQ ID NO:110 966TGGAGACAGAGTGAGGGTCT −18.5 −24.6 73.8 −4.8 −1.2 −5.9 SEQ ID NO:111 1052ATTGGTATGCTTTTTTTTTT −18.5 −19.8 62.1 −1.2 0 −3.6 SEQ ID NO:112 1064CGTGTCTGGTTCATTGGTAT −18.5 −24.3 72.2 −5.8 0 −2.7 SEQ ID NO:113 159AGGAGGGAAGAGATTAGAAC −18.4 −19.2 59.2 −0.6 0 −1.4 SEQ ID NO:114 686TGACGAGAGAAGAAGACACT −18.4 −19.2 57.8 −0.6 0 −3.5 SEQ ID NO:115 1047TATGCTTTTTTTTTTTTGTC −18.4 −19.1 61.3 −0.4 0 −3.6 SEQ ID NO:116 141ACTTTCATCGCAACTGTCGG −18.3 −24.1 68.4 −5.8 0 −4.7 SEQ ID NO:117 683CGAGAGAAGAAGACACTAGA −18.3 −18.7 56.9 0 0 −4.5 SEQ ID NO:118 895TAAAATGGGTCATCTGGTTG −18.3 −20.1 60.9 −1.8 0 −2.9 SEQ ID NO:119 3AGCGTTCCCATTTGAGGGCG −18.2 −28.8 77.2 −9 −1.5 −9.2 SEQ ID NO:120 157GAGGGAAGAGATTAGAACTT −18.2 −19 58.7 −0.6 0 −2.6 SEQ ID NO:121 563TGTCTGGTAGGTGTGCTCAC −18.2 −26.2 79.5 −6.7 −1.2 −3.3 SEQ ID NO:122 901ATAGGGTAAAATGGGTCATC −18.2 −20 61 −1.8 0 −2.9 SEQ ID NO:123 155GGGAAGAGATTAGAACTTTC −18.1 −18.9 58.9 −0.6 0 −3.2 SEQ ID NO:124 964GAGACAGAGTGAGGGTCTTG −18.1 −23.5 71.3 −3.9 −1.4 −5.5 SEQ ID NO:125 716CCTGGGTAAGGGGAGGGCAC −18 −28.8 79.5 −10 −0.6 −5.2 SEQ ID NO:126 934GTATGTGTAGAATCTGGATT −18 −20.1 62.6 −2.1 0 −6.7 SEQ ID NO:127 233CCCTGTGGCCTCTGGCGACC −17.9 −33.9 87.2 −16 1.9 −7.2 SEQ ID NO:128 684ACGAGAGAAGAAGACACTAG −17.9 −18.3 56.2 0 0 −4 SEQ ID NO:129 935AGTATGTGTAGAATCTGGAT −17.9 −20 62.5 −2.1 0 −4.5 SEQ ID NO:130 65ATCCCTGGGGATGACTCAGG −17.8 −27.5 76.7 −6.9 −2.8 −11.1 SEQ ID NO:131 224CTCTGGCGACCCCTGGATTC −17.8 −30 80 −11.5 −0.4 −5.2 SEQ ID NO:132 271GCCTTCCTGGAGCCATCTCC −17.8 −32.1 87.2 −11.9 −2.4 −6.8 SEQ ID NO:133 399TGTCTTGTTTTCTTCACATT −17.8 −21.6 67.5 −3.8 0 −2.7 SEQ ID NO:134 485GCAGAGCAAAGCTTCTTAGC −17.8 −23.9 70.4 −4.8 −1.2 −7.7 SEQ ID NO:135 713GGGTAAGGGGAGGGCACAGG −17.8 −27.8 78.2 −10 0 −4 SEQ ID NO:136 905GTGAATAGGGTAAAATGGGT −17.8 −19.6 59.2 −1.8 0 −1.2 SEQ ID NO:137 1062TGTCTGGTTCATTGGTATGC −17.8 −24.1 73.1 −5.8 −0.1 −2.6 SEQ ID NO:138 151AGAGATTAGAACTTTCATCG −17.7 −18.5 57.7 −0.6 0 −4.2 SEQ ID NO:139 156AGGGAAGAGATTAGAACTTT −17.7 −18.5 57.7 −0.6 0 −3.2 SEQ ID NO:140 232CCTGTGGCCTCTGGCGACCC −17.7 −33.9 87.2 −16.2 1.9 −6.5 SEQ ID NO:141 903GAATAGGGTAAAATGGGTCA −17.7 −19.5 58.9 −1.8 0 −2.1 SEQ ID NO:142 959AGAGTGAGGGTCTTGGTGGG −17.7 −26.2 78.3 −8.5 0 −2.5 SEQ ID NO:143 1063GTGTCTGGTTCATTGGTATG −17.7 −23.5 72.2 −5.8 0 −2.7 SEQ ID NO:144 139TTTCATCGCAACTGTCGGTG −17.6 −24.2 69 −5.8 −0.6 −6.7 SEQ ID NO:145 223TCTGGCGACCCCTGGATTCA −17.6 −29.8 79.1 −11.5 −0.4 −5.2 SEQ ID NO:146 428GCTTGTTTGGCTTTCTGTGG −17.6 −26 77.3 −8.4 0 −3.7 SEQ ID NO:147 486GGCAGAGCAAAGCTTCTTAG −17.6 −23.3 68.7 −4.8 −0.7 −7.7 SEQ ID NO:148 1060TCTGGTTCATTGGTATGCTT −17.6 −23.9 72.2 −5.8 −0.1 −3.6 SEQ ID NO:149 487AGGCAGAGCAAAGCTTCTTA −17.5 −23.3 68.7 −4.8 −0.9 −7.7 SEQ ID NO:150 608ATTTAGGGGTGGGTACAGTG −17.5 −24.1 72.2 −5.9 −0.4 −5.2 SEQ ID NO:151 680GAGAAGAAGACACTAGAGAG −17.5 −17.9 56.4 0 0 −4.5 SEQ ID NO:152 681AGAGAAGAAGACACTAGAGA −17.5 −17.9 56.4 0 0 −4.5 SEQ ID NO:153 682GAGAGAAGAAGACACTAGAG −17.5 −17.9 56.4 0 0 −4.5 SEQ ID NO:154 981GAACAAGTAGGCCAATGGAG −17.5 −21.8 63.2 −3.8 0 −7.7 SEQ ID NO:155 982TGAACAAGTAGGCCAATGGA −17.5 −21.8 62.9 −3.8 0 −7.7 SEQ ID NO:156 1053CATTGGTATGCTTTTTTTTT −17.5 −20.4 63 −2.9 0 −3.6 SEQ ID NO:157 163CAGGAGGAGGGAAGAGATTA −17.4 −21.6 64.6 −4.2 0 −1.5 SEQ ID NO:158 220GGCGACCCCTGGATTCAGGC −17.3 −31.5 82.7 −11.5 −2.7 −11 SEQ ID NO:159 862CCCATTTGAAGGAAACAATT −17.3 −19.5 57 −2.2 0 −3.4 SEQ ID NO:160 1059CTGGTTCATTGGTATGCTTT −17.3 −23.6 70.8 −5.8 −0.1 −3.6 SEQ ID NO:161 131CAACTGTCGGTGCAGCTGTA −17.2 −26 74.1 −7.3 −1.3 −9.9 SEQ ID NO:162 936AAGTATGTGTAGAATCTGGA −17.2 −19.3 60.3 −2.1 0 −4 SEQ ID NO:163 961ACAGAGTGAGGGTCTTGGTG −17.2 −24.7 74.5 −7.5 0 −2.8 SEQ ID NO:164 230TGTGGCCTCTGGCGACCCCT −17.1 −33.9 87.2 −16.8 1.9 −7.6 SEQ ID NO:165 902AATAGGGTAAAATGGGTCAT −17.1 −18.9 57.6 −1.8 0 −2.9 SEQ ID NO:166 972GGCCAATGGAGACAGAGTGA −17.1 −24.7 70.4 −6.7 −0.8 −8.5 SEQ ID NO:167 219GCGACCCCTGGATTCAGGCT −17 −31.2 82.1 −11.5 −2.7 −9.6 SEQ ID NO:168 222CTGGCGACCCCTGGATTCAG −17 −29.4 77.8 −11.5 −0.7 −6.6 SEQ ID NO:169 554GGTGTGCTCACTGTCTTCTT −17 −26.5 80.4 −7.5 −2 −4.2 SEQ ID NO:170 904TGAATAGGGTAAAATGGGTC −17 −18.8 57.6 −1.8 0 −1.7 SEQ ID NO:171 1058TGGTTCATTGGTATGCTTTT −17 −22.8 69.1 −5.8 0.5 −3.6 SEQ ID NO:172 150GAGATTAGAACTTTCATCGC −16.9 −20.3 61.6 −3.4 0 −4.2 SEQ ID NO:173 154GGAAGAGATTAGAACTTTCA −16.9 −18.4 57.6 −0.6 −0.4 −4.6 SEQ ID NO:174 164ACAGGAGGAGGGAAGAGATT −16.9 −22.1 65.7 −5.2 0 −1.3 SEQ ID NO:175 555AGGTGTGCTCACTGTCTTCT −16.0 −26.4 80.3 −7.5 −2 −4.2 SEQ ID NO:176 619GCACTGGAATGATTTAGGGG −16.9 −22.8 66.5 −5.9 0 −3.4 SEQ ID NO:177 967ATGGAGACAGAGTGAGGGTC −16.9 −23.7 71.6 −5.9 −0.8 −5.2 SEQ ID NO:178 983ATGAACAAGTAGGCCAATGG −16.9 −21.2 61.6 −3.8 0 −7.7 SEQ ID NO:179 1066ACCGTGTCTGGTTCATTGGT −16.9 −26.8 77.3 −9 −0.7 −4.7 SEQ ID NO:180 610TGATTTAGGGGTGGGTACAG −16.6 −23.5 70.1 −6.2 −0.4 −5.2 SEQ ID NO:181 679AGAAGAAGACACTAGAGAGA −16.6 −17.9 56.4 −1.2 0 −4.5 SEQ ID NO:182 906AGTGAATAGGGTAAAATGGG −16.6 −18.4 56.5 −1.8 0 −1.2 SEQ ID NO:183 1057GGTTCATTGGTATGCTTTTT −16.6 −22.9 69.7 −5.8 −0.1 −3.6 SEQ ID NO:184 142AACTTTCATCGCAACTGTCG −16.4 −22.2 63.8 −5.8 0 −4.1 SEQ ID NO:185 153GAAGAGATTAGAACTTTCAT −16.4 −17.2 55 −0.6 0 −4.6 SEQ ID NO:186 177ATTAGTGGCAGCAACAGGAG −16.4 −23.2 68.4 −6.8 0 −2.4 SEQ ID NO:187 687CTGACGAGAGAAGAAGACAC −16.4 −19.2 57.8 −2.8 0 −3.5 SEQ ID NO:188 973AGGCCAATGGAGACAGAGTG −16.4 −24.1 69.4 −6.7 −0.8 −9.2 SEQ ID NO:189 149AGATTAGAACTTTCATCGCA −16.3 −20.4 61.5 −4.1 0 −4.2 SEQ ID NO:190 231CTGTGGCCTCTGGCGACCCC −16.3 −33.9 87.2 −17.6 1.9 −7.3 SEQ ID NO:191 237CGGTCCCTGTGGCCTCTGGC −16.3 −33.9 90.1 −16 −1.5 −7.2 SEQ ID NO:192 559TGGTAGGTGTGCTCACTGTC −16.3 −26.2 79.5 −7.9 −2 −4.2 SEQ ID NO:193 616CTGGAATGATTTAGGGGTGG −16.3 −22.5 66.2 −6.2 0 −2.3 SEQ ID NO:194 618CACTGGAATGATTTAGGGGT −16.3 −22.2 65.5 −5.9 0 −2.3 SEQ ID NO:195 932ATGTGTAGAATCTGGATTCA −16.3 −20.3 62.8 −2.1 −1.7 −11 SEQ ID NO:196 937TAAGTATGTGTAGAATCTGG −16.3 −18.4 58.4 −2.1 0 −4 SEQ ID NO:197 984GATGAACAAGTAGGCCAATG −16.3 −20.6 60.4 −3.8 0 −7.7 SEQ ID NO:198 985AGATGAACAAGTAGGCCAAT −16.3 −20.6 60.7 −3.8 0 −7.7 SEQ ID NO:199 1054TCATTGGTATGCTTTTTTTT −16.3 −20.7 −3.9 −0.1 −3.6 SEQ ID NO:200 99AATATAATGGAAGGTTCCCT −16.2 −20.9 61.3 −3.7 −0.8 −7.1 SEQ ID NO:201 143GAACTTTCATCGCAACTGTC −16.2 −22 64.8 −5.8 0 −3.6 SEQ ID NO:202 152AAGAGATTAGAACTTTCATC −16.2 −17 55 −0.6 0 −4.6 SEQ ID NO:203 217GACCCCTGGATTCAGGCTGC −16.2 −30.4 82.4 −11.5 −2.7 −9.6 SEQ ID NO:204 429TGCTTGTTTGGCTTTCTGTG −16.2 −24.8 74.3 −7.7 −0.7 −3.7 SEQ ID NO:205 430ATGCTTGTTTGGCTTTCTGT −16.2 −24.8 74.4 −7.7 −0.7 −3.7 SEQ ID NO:206 718AGCCTGGGTAAGGGGAGGGC −16.2 −29.7 82.6 −12.1 −1.3 −6.7 SEQ ID NO:207 933TATGTGTAGAATCTGGATTC −16.2 −19.3 60.9 −2.1 −0.6 −9.7 SEQ ID NO:208 971GCCAATGGAGACAGAGTGAG −16.2 −23.5 68.1 −6.7 −0.3 −6.3 SEQ ID NO:209 270CCTTCCTGGAGCCATCTCCT −16.1 −31.2 84.7 −11.9 −3.2 −7.4 SEQ ID NO:210 398GTCTTGTTTTCTTCACATTG −16.1 −21.6 67.5 −5.5 0 −2.7 SEQ ID NO:211 558GGTAGGTGTGCTCACTGTCT −16.1 −27.1 81.9 −9.7 −1.2 −3.4 SEQ ID NO:212 886TCATCTGGTTGTGAATTGGC −16.1 −23.1 69.1 −7 0 −3.1 SEQ ID NO:213 974TAGGCCAATGGAGACAGAGT −16.1 −23.8 69 −6.7 −0.8 −9.2 SEQ ID NO:214 480GCAAAGCTTCTTAGCTGACA −16 −23.2 68 −4.8 −2.4 −8.1 SEQ ID NO:215 569GAAGAGTGTCTGGTAGGTGT −16 −23.9 73.5 −7.9 0 −2.9 SEQ ID NO:216 604AGGGGTGGGTACAGTGGGAG −16 −27.2 79.4 −10.5 −0.4 −5.2 SEQ ID NO:217 100GAATATAATGGAAGGTTCCC −15.9 −20.6 60.7 −3.7 −0.8 −7.1 SEQ ID NO:218 609GATTTAGGGGTGGGTACAGT −15.9 −24.7 73.9 −8.1 −0.4 −5.2 SEQ ID NO:219 130AACTGTCGGTGCAGCTGTAA −15.8 −24.6 70.6 −7.3 −1.3 −9.9 SEQ ID NO:220 144AGAACTTTCATCGCAACTGT −15.8 −21.6 63.6 −5.8 0 −4.2 SEQ ID NO:221 481AGCAAAGCTTCTTAGCTGAC −15.8 −22.5 67.1 −4.8 −1.9 −8.8 SEQ ID NO:222 863CCCCATTTGAAGGAAACAAT −15.8 −21.4 60.1 −5.6 0 −3.4 SEQ ID NO:223 103GAAGAATATAATGGAAGGTT −15.7 −16.1 51.7 0 0 −2.5 SEQ ID NO:224 218CGACCCCTGGATTCAGGCTG −15.7 −29.4 77.8 −11.5 −2.2 −9.1 SEQ ID NO:225 221TGGCGACCCCTGGATTCAGG −15.7 −29.7 78.4 −11.5 −2.5 −11 SEQ ID NO:226 939GATAAGTATGTGTAGAATCT −15.7 −17.8 57.1 −2.1 0 −3.6 SEQ ID NO:227 944GTGGGGATAAGTATGTGTAG −15.7 −21.4 65.7 −5.7 0 −1.8 SEQ ID NO:228 993TGAGTGAAAGATGAACAAGT −15.7 −16.9 53.4 −1.1 0 −2.9 SEQ ID NO:229 1002TTTGTCGAATGAGTGAAAGA −15.7 −18.1 55.9 −2.4 0 −5 SEQ ID NO:230 63CCCTGGGGATGACTCAGGTC −15.6 −28.7 80.3 −10 −3.1 −9 SEQ ID NO:231 104TGAAGAATATAATGGAAGGT −15.6 −16 51.4 0 0 −2.7 SEQ ID NO:232 133CGCAACTGTCGGTGCAGCTG −15.6 −27.7 75.4 −10.5 −1.6 −8.3 SEQ ID NO:233 1001TTGTCGAATGAGTGAAAGAT −15.6 −18 55.6 2.4 0 −5 SEQ ID NO:234 717GCCTGGGTAAGGGGAGGGCA −15.5 −30.4 83.3 −13.4 −1.4 −7 SEQ ID NO:235 990GTGAAAGATGAACAAGTAGG −15.5 −17.2 54.1 −1.7 0 −2.9 SEQ ID NO:236 1000TGTCGAATGAGTGAAAGATG 15.5 −17.9 55.3 −2.4 0 −5 SEQ ID NO:237 178CATTAGTGGCAGCAACAGGA −15.4 −23.9 69.3 −8.5 0 −1.6 SEQ ID NO:238 236GGTCCCTGTGGCCTCTGGCG −15.4 −33.9 90.1 −16 −2.5 −7.7 SEQ ID NO:239 475GCTTCTTAGCTGACATTGTT −15.4 −23.5 70.9 −6.8 −1.2 −7.2 SEQ ID NO:240 980AACAAGTAGGCCAATGGAGA −15.4 −21.8 63.2 −5.9 0 −7.7 SEQ ID NO:241 992GAGTGAAAGATGAACAAGTA −15.4 −16.6 52.9 −1.1 0 −2.9 SEQ ID NO:242 94AATGGAAGGTTCCCTGCTGG −15.3 −26.1 72.6 −9.9 −0.8 −7.1 SEQ ID NO:243 488AAGGCAGAGCAAAGCTTCTT −15.3 −22.9 67 −6.6 −0.9 −7.7 SEQ ID NO:244 1055TTCATTGGTATGCTTTTTTT −15.3 −20.7 64.2 −4.9 −0.1 −3.6 SEQ ID NO:245 90GAAGGTTCCCTGCTGGAGGC −15.2 −29.2 81.2 −13.1 −0.8 −7.8 SEQ ID NO:246 98ATATAATGGAAGGTTCCCTG −15.2 −21.6 63.2 −5.5 −0.8 −7.1 SEQ ID NO:247 484CAGAGCAAAGCTTCTTAGCT −15.2 −23 68.1 −5.6 −2.2 −8.5 SEQ ID NO:248 603GGGGTGGGTACAGTGGGAGA −15.1 −27.8 80.5 −12 −0.4 −5.2 SEQ ID NO:249 938ATAAGTATGTGTAGAATCTG −15.1 −17.2 55.7 −2.1 0 −4 SEQ ID NO:250 1003ATTTGTCGAATGAGTGAAAG −15.1 −17.5 54.7 −2.4 0 −4.5 SEQ ID NO:251 474CTTCTTAGCTGACATTGTTT −15 −21.8 66.8 −6.8 0 −5.3 SEQ ID NO:252 678GAAGAAGACACTAGAGAGAG −15 −17.9 56.4 −2.9 0 −4.5 SEQ ID NO:253 975GTAGGCCAATGGAGACAGAG −15 −23.8 69 −7.8 −0.8 −9.2 SEQ ID NO:254 28GTGGTCTATGCTTTAGTCCC −14.9 −26.8 79.2 −11.9 0 −4 SEQ ID NO:255 66GATCCCTGGGGATGACTCAG −14.9 −26.9 75.5 −10 −1.4 −11.9 SEQ ID NO:256 482GAGCAAAGCTTCTTAGCTGA −14.9 −22.9 67.8 −5.6 −2.4 −8.8 SEQ ID NO:257 847CAATTTTGATCTGTGACATT −14.9 −19 58.8 −4.1 0 −4.9 SEQ ID NO:258 134TCGCAACTGTCGGTGCAGCT −14.8 −28.1 77.2 −11.7 −1.6 −8.4 SEQ ID NO:259 620AGCACTGGAATGATTTAGGG −14.8 −21.6 64.1 −6.8 0 −4.1 SEQ ID NO:260 858TTTGAAGGAAACAATTTTGA −14.8 −15.6 50.5 −0.6 0 −4.4 SEQ ID NO:261 991AGTGAAAGATGAACAAGTAG −14.8 −16 51.8 −1.1 0 −2.9 SEQ ID NO:262 1046ATGCTTTTTTTTTTTTGTCC −14.8 −21.4 65.9 −6.6 0 −3.6 SEQ ID NO:263 1069AAGACCGTGTCTGGTTCATT −14.8 −24.3 70.5 −8.1 −1.3 −8.3 SEQ ID NO:264 1077TCTTTAATAAGACCGTGTCT −14.8 −20.8 62.2 −4.8 −1.1 −8 SEQ ID NO:265 483AGAGCAAAGCTTCTTAGCTG −14.7 −22.3 66.7 −5.2 −2.4 −8.8 SEQ ID NO:266 885CATCTGGTTGTGAATTGGCA −14.7 −23.4 68.7 −8.7 0 −4 SEQ ID NO:267 91GGAAGGTTCCCTGCTGGAGG −14.6 −28.6 79.4 −13.1 −0.8 −6.8 SEQ ID NO:268 102AAGAATATAATGGAAGGTTC −14.6 −15.9 51.7 −1.2 0 −3.3 SEQ ID NO:269 165AACAGGAGGAGGGAAGAGAT −14.6 −21.3 63.2 −6.7 0 −1.1 SEQ ID NO:270 476AGCTTCTTAGCTGACATTGT −14.6 −23.4 70.8 −6.8 −2 −7.7 SEQ ID NO:271 711GTAAGGGGAGGGCACAGGCT −14.6 −28.1 79.4 −12.1 −1.3 −4 SEQ ID NO:272 994ATGAGTGAAAGATGAACAAG −14.5 −15.7 50.7 −1.1 0 −2.9 SEQ ID NO:273 968AATGGAGACAGAGTGAGGGT −14.4 −22.6 67.5 −7.3 −0.8 −3.7 SEQ ID NO:274 1070TAAGACCGTGTCTGGTTCAT −14.4 −23.9 69.5 −8.1 −1.3 −8.3 SEQ ID NO:275 1071ATAAGACCGTGTCTGGTTCA −14.4 −23.9 69.5 −8.1 −1.3 −8.3 SEQ ID NO:276 145TAGAACTTTCATCGCAACTG −14.3 −20.1 60 −5.8 0 −4.2 SEQ ID NO:277 431AATGCTTGTTTGGCTTTCTG −14.3 −22.9 68.4 −7.7 −0.7 −3.7 SEQ ID NO:278 712GGTAAGGGGAGGGCACAGGC −14.3 −28.4 80 −13.4 −0.5 −4 SEQ ID NO:279 4CAGCGTTCCCATTTGAGGGC −14.2 −28.7 78.6 −13.2 −1.2 −9.2 SEQ ID NO:280 101AGAATATAATGGAAGGTTCC −14.2 −18.6 57.2 −3.7 −0.4 −6.7 SEQ ID NO:281 844TTTTGATCTGTGACATTTAA −14.2 −18.1 57.3 −3.9 0 −4.9 SEQ ID NO:282 907CAGTGAATAGGGTAAAATGG −14.2 −17.9 55.3 −3.7 0 −3.1 SEQ ID NO:283 89AAGGTTCCCTGCTGGAGGCT −14.1 −29.5 81.8 −14 −1.3 −8 SEQ ID NO:284 93ATGGAAGGTTCCCTGCTGGA −14.1 −27.4 76.3 −12.4 −0.8 −7.1 SEQ ID NO:285 688ACTGACGAGAGAAGAAGACA −14.1 −19.2 57.8 −5.1 0 −3.4 SEQ ID NO:286 869GGCAGACCCCATTTGAAGGA −14.1 −27.1 73.5 −13 0 −4 SEQ ID NO:287 979ACAAGTAGGCCAATGGAGAC −14.1 −22.7 65.8 −8.1 0 −7.7 SEQ ID NO:288 491ACAAAGGCAGAGCAAAGCTT −13.9 −21.7 62.9 −6.8 −0.9 −7.5 SEQ ID NO:289 676AGAAGACACTAGAGAGAGCA −13.9 −20.5 62.6 −6.6 0 −4.5 SEQ ID NO:290 95TAATGGAAGGTTCCCTGCTG −13.8 −24.6 69.6 −9.9 −0.8 −7.1 SEQ ID NO:291 269CTTCCTGGAGCCATCTCCTA −13.8 −28.9 80.6 −11.9 −3.2 −7.4 SEQ ID NO:292 489AAAGGCAGAGCAAAGCTTCT −13.8 −22.1 64.5 −7.3 −0.9 −7.7 SEQ ID NO:293 864ACCCCATTTGAAGGAAACAA −13.8 −21.6 60.5 −7.8 0 −3.4 SEQ ID NO:294 1078ATCTTTAATAAGACCGTGTC −13.8 −19.9 60.3 −4.8 −1.2 −6.8 SEQ ID NO:295 148GATTAGAACTTTCATCGCAA −13.7 −19.7 59.3 −6 0 −3.6 SEQ ID NO:296 394TGTTTTCTTCACATTGCCCT −13.7 −25.7 74.2 −12 0 −3 SEQ ID NO:297 719AAGCCTGGGTAAGGGGAGGG −13.7 −27.2 75.7 −12.1 −1.3 −5.2 SEQ ID NO:298 913AGTCTGCAGTGAATAGGGTA −13.7 −23.1 70.1 −8.8 0 −8.6 SEQ ID NO:299 105TTGAAGAATATAATGGAAGG −13.6 −14.9 49.1 −1.2 0 −2.7 SEQ ID NO:300 213CCTGGATTCAGGCTGCTAGA −13.6 −26.8 76.5 −11 −2.2 −9.4 SEQ ID NO:301 216ACCCCTGGATTCAGGCTGCT −13.6 −30.7 83 −14.4 −2.7 −9.6 SEQ ID NO:302 272CGCCTTCCTGGAGCCATCTC −13.6 −30.9 83.1 −16.4 −0.7 −6.7 SEQ ID NO:303 363CAGGGGCACTGCTTCTTTGG −13.6 −27.4 78.2 −13.1 −0.5 −6 SEQ ID NO:304 368GATCACAGGGGCACTGCTTC −13.6 −27 77.8 −12.7 −0.5 −7.7 SEQ ID NO:305 492TACAAAGGCAGAGCAAAGCT −13.6 −21.3 62.1 −6.8 −0.7 −5.7 SEQ ID NO:306 557GTAGGTGTGCTCACTGTCTT −13.6 −26 79.4 −10.4 −2 −4.2 SEQ ID NO:307 677AAGAAGACACTAGAGAGAGC −13.6 −19.1 59.2 −5.5 0 −4.5 SEQ ID NO:308 998TCGAATGAGTGAAAGATGAA −13.6 −16.6 52.1 −3 0 −4.2 SEQ ID NO:309 1045TGCTTTTTTTTTTTTGTCCC −13.6 −23.4 69.9 −9.8 0 −3.6 SEQ ID NO:310 1056GTTCATTGGTATGCTTTTTT −13.6 −21.8 67.3 −7.7 −0.1 −3.6 SEQ ID NO:311 88AGGTTCCCTGCTGGAGGCTC −13.5 −30.6 86.6 −15.9 −1.1 −8 SEQ ID NO:312 128CTGTCGGTGCAGCTGTAAGT −13.5 −26.3 76.2 −12 −0.4 −8.9 SEQ ID NO:313 188TGGACATCAGCATTAGTGGC −13.5 −24.3 71.7 −10.8 0 −4.1 SEQ ID NO:314 274GCCGCCTTCCTGGAGCCATC −13.5 −33.4 87 −19.2 −0.4 −6.7 SEQ ID NO:315 289GCACTCACATTCTTGGCCGC −13.5 −28.7 78.9 −14.7 0 −7.6 SEQ ID NO:316 92TGGAAGGTTCCCTGCTGGAG −13.4 −27.4 76.6 −13.1 −0.8 −7.1 SEQ ID NO:317 601GGTGGGTACAGTGGGAGAGT −13.4 −26.6 79.1 −12.5 −0.4 −4.6 SEQ ID NO:318 602GGGTGGGTACAGTGGGAGAG −13.4 −26.6 78.1 −12.5 −0.4 −5.2 SEQ ID NO:319 617ACTGGAATGATTTAGGGGTG −13.4 −21.5 64.2 −8.1 0 −2.3 SEQ ID NO:320 843TTTGATCTGTGACATTTAAA −13.4 −17.3 55 −3.9 0 −4.9 SEQ ID NO:321 853AGGAAACAATTTTGATCTGT −13.3 −18 56.1 −4.7 0 −5.8 SEQ ID NO:322 67TGATCCCTGGGGATGACTCA −13.2 −26.9 75 −11.7 −1.4 −11.9 SEQ ID NO:323 179GCATTAGTGGCAGCAACAGG −13.2 −25.1 72.2 −11.9 0 −2.4 SEQ ID NO:324 366TCACAGGGGCACTGCTTCTT −13.2 −27.4 78.9 −12.7 −1.4 −6.5 SEQ ID NO:325 397TCTTGTTTTCTTCACATTGC −13.2 −22.2 68.6 −9 0 −2.7 SEQ ID NO:326 857TTGAAGGAAACAATTTTGAT −13.2 −15.5 50.2 −2.3 0 −4.4 SEQ ID NO:327 62CCTGGGGATGACTCAGGTCA −13.1 −27.4 77.8 −11.7 −2.6 −8 SEQ ID NO:328 97TATAATGGAAGGTTCCCTGC −13.1 −23.4 67.2 −9.4 −0.8 −7.1 SEQ ID NO:329 367ATCACAGGGGCACTGCTTCT −13.1 −27.3 78.5 −12.7 −1.4 −6.5 SEQ ID NO:330 710TAAGGGGAGGGCACAGGCTA −13.1 −26.6 75.2 −12.1 −1.3 −4 SEQ ID NO:331 882CTGGTTGTGAATTGGCAGAC −13.1 −23.1 68.1 −10 0 −4 SEQ ID NO:332 1079TATCTTTAATAAGACCGTGT −13.1 −19.2 58.4 −4.8 −1.2 −6 SEQ ID NO:333 393GTTTTCTTCACATTGCCCTT −13 −25.8 74.7 −12.8 0 −3 SEQ ID NO:334 570AGAAGAGTGTCTGGTAGGTG −13 −22.7 70 −9.7 0 −2.9 SEQ ID NO:335 859ATTTGAAGGAAACAATTTTG −13 −15 49.3 −2 0 −3.9 SEQ ID NO:336 914CAGTCTGCAGTGAATAGGGT −13 −24.1 71.9 −10.5 0 −8.6 SEQ ID NO:337 395TTGTTTTCTTCACATTGCCC −12.9 −24.9 72.6 −12 0 −3 SEQ ID NO:338 931TGTGTAGAATCTGGATTCAG −12.9 −20.3 63 −5.6 −1.7 −11 SEQ ID NO:339 976AGTAGGCCAATGGAGACAGA −12.9 −23.8 69 −9.9 −0.8 −9.2 SEQ ID NO:340 1004GATTTGTCGAATGAGTGAAA −12.9 −18.1 55.8 −5.2 0 −5 SEQ ID NO:341 1067GACCGTGTCTGGTTCATTGG −12.9 −26.2 75.1 −11.9 −1.3 −7.8 SEQ ID NO:342 129ACTGTCGGTGCAGCTGTAAG −12.8 −25.3 73.3 −11 −1.3 −9.9 SEQ ID NO:343 845ATTTTGATCTGTGACATTTA −12.8 −18.8 59.3 −6 0 −4.2 SEQ ID NO:344 852GGAAACAATTTTGATCTGTG −12.8 −18 55.9 −4.7 −0.2 −5.8 SEQ ID NO:345 870TGGCAGACCCCATTTGAAGG −12.8 −26.5 72.1 −13 −0.5 −4.4 SEQ ID NO:346 988GAAAGATGAACAAGTAGGCC −12.8 −19.8 58.9 −7 0 −6.4 SEQ ID NO:347 573AGAAGAAGAGTGTCTGGTAG −12.7 −20.2 63.1 −7.5 0 −2.9 SEQ ID NO:348 930GTGTAGAATCTGGATTCAGT −12.7 −21.5 66.5 −7.4 −1.1 −10.2 SEQ ID NO:349 1044GCTTTTTTTTTTTTGTCCCA −12.7 −24.1 71.2 −11.4 0 −2.8 SEQ ID NO:350 75GAGGCTCCTGATCCCTGGGG −12.6 −31.3 84.9 −18.1 −0.2 −8.2 SEQ ID NO:351 238TCGGTCCCTGTGGC2CTGG −12.6 −32.5 87.6 −18.3 −1.5 −7.2 SEQ ID NO:352 795TCCTGATTGCATTT3AGGTT −12.6 −22.2 66 −9.1 −0.1 −5.4 SEQ ID NO:353 796TTCCTGATTGCATT4AAGGT −12.6 −22.2 66 −9.1 −0.1 −5.4 SEQ ID NO:354 842TTGATCTGTGACAT5TAAAA −12.6 −16.5 52.9 −3.9 0 −5 SEQ ID NO:355 865GACCCCATTTGAAG6AAACA −12.6 −22.9 63.5 −10.3 0 −3.4 SEQ ID NO:356 943TGGGGATAAGTATGTGTAGA −12.6 −20.8 63.8 −8.2 0 −1.6 SEQ ID NO:357 989TGAAAGATGAACAAGTAGGC −12.6 −17.8 55.2 −5.2 0 −2.9 SEQ ID NO:358 999GTCGAATGAGTGAAAGATGA −12.6 −18.5 56.6 −5.9 0 −5 SEQ ID NO:359 9CAGGCCAGCGTTCCCATTTG −12.5 −29.6 79.2 −16.6 0 −7.7 SEQ ID NO:360 215CCCCTGGATTCAGGCTGCTA −12.5 −30.2 81.8 −15 −2.7 −9.6 SEQ ID NO:361 8AGGCCAGCGTTCCCATTTGA −12.4 −29.5 79.5 −16.6 0 −7.7 SEQ ID NO:362 96ATAATGGAAGGTTCCCTGCT −12.4 −24.6 69.7 −11.5 −0.4 −6.4 SEQ ID NO:363 369TGATCACAGGGGCACTGCTT −12.4 −26.6 75.8 −12.7 −1.4 −7.5 SEQ ID NO:364 391TTTCTTCACATTGCCCTTGA −12.4 −25.1 72.1 −12.7 0 −3 SEQ ID NO:365 479CAAAGCTTCTTAGCTGACAT −12.4 −21.4 63.8 −6.6 −2.4 −7 SEQ ID NO:366 522TTAATTGGAAGAGTGGGCGC −12.4 −22.9 65.9 −10.5 0 −7.2 SEQ ID NO:367 794CCTGATTGCATTTAAGGTTA −12.4 −21.5 63.9 −9.1 0 −5.1 SEQ ID NO:368 27TGGTCTATGCTTTAGTCCCA −12.3 −26.3 76.6 −13 −0.9 −5.7 SEQ ID NO:369 370ATGATCACAGGGGCACTGCT −12.3 −26.5 75.4 −12.7 −1.4 −8.7 SEQ ID NO:370 551GTGCTCACTGTCTTCTTGGC −12.3 −27.1 81.3 −14.8 0 −4.7 SEQ ID NO:371 912GTCTGCAGTGAATAGGGTAA −12.3 −22.4 67.4 −9.5 0 −8.6 SEQ ID NO:372 74AGGCTCCTGATCCCTGGGGA −12.2 −31.3 84.9 −18.1 −0.2 −9.9 SEQ ID NO:373 110GTTGCTTGAAGAATATAATG −12.2 −16.6 53.1 −4.4 0 −3.6 SEQ ID NO:374 111AGTTGCTTGAAGAATATAAT −12.2 −16.6 53.3 −4.4 0 −3.6 SEQ ID NO:375 187GGACATCAGCATTAGTGGCA −12.2 −25 73 −11.9 −0.8 −4.1 SEQ ID NO:376 234TCCCTGTGGCCTCTGGCGAC −12.2 −32.3 85.8 −17.6 −2.5 −8.6 SEQ ID NO:377 521TAATTGGAAGAGTGGGCGCT −12.2 −23.7 67.4 −11 −0.1 −8.1 SEQ ID NO:378 689GACTGACGAGAGAAGAAGAC −12.2 −19.1 57.8 −6.9 0 −3.5 SEQ ID NO:379 868GCAGACCCCATTTGAAGGAA −12.2 −25.2 69 −13 0 −3.4 SEQ ID NO:380 878TTGTGAATTGGCAGACCCCA −12.2 −26.5 72.4 −13.6 −0.5 −4 SEQ ID NO:381 969CAATGGAGACAGAGTGAGGG −12.2 −22.1 65.4 −9 −0.8 −4.5 SEQ ID NO:382 1076CTTTAATAAGACCGTGTCTG −12.2 −20.4 60.8 −6.8 −1.3 −8.3 SEQ ID NO:383 275GGCCGCCTTCCTGGAGCCAT −12.1 −34.2 87.6 −19.2 −2.9 −9.6 SEQ ID NO:384 364ACAGGGGCACTGCTTCTTTG −12.1 −26.4 76.2 −12.8 −1.4 −6.5 SEQ ID NO:385 675GAAGACACTAGAGAGAGCAA −12.1 −19.8 60.3 −7.7 0 −4.5 SEQ ID NO:386 690AGACTGACGAGAGAAGAAGA −12.1 −18.9 57.5 −6.8 0 −3.5 SEQ ID NO:387 877TGTGAATTGGCAGACCCCAT −12.1 −26.4 72.1 −13.6 −0.5 −4 SEQ ID NO:388 940GGATAAGTATGTGTAGAATC −12.1 −18.1 57.8 −6 0 −2.7 SEQ ID NO:389 549GCTCACTGTCTTCTTGGCTG −12 −26.8 79.5 −14.8 0 −3.7 SEQ ID NO:390 553GTGTGCTCACTGTCTTCTTG −12 −25.3 77.2 −12 −1.2 −3.3 SEQ ID NO:391 978CAAGTAGGCCAATGGAGACA −12 −23.2 66.4 −10.3 −0.6 −8.9 SEQ ID NO:392 1080TTATCTTTAATAAGACCGTG −12 −18.1 55.9 −4.8 −1.2 −6 SEQ ID NO:393 1081ATTATCTTTAATAAGACCGT −12 −18.1 55.9 −4.8 −1.2 −6 SEQ ID NO:394 113TAAGTTGCTTGAAGAATATA −11.9 −16.3 52.7 −4.4 0 −4.3 SEQ ID NO:395 273CCGCCTTCCTGGAGCCATCT −11.9 −32.5 84.6 −20 −0.3 −6.7 SEQ ID NO:396 874GAATTGGCAGACCCCATTTG −11.9 −25.4 69.8 −13 −0.2 −4 SEQ ID NO:397 520AATTGGAAGAGTGGGCGCTC −11.8 −24.4 69.5 11 −1.6 −8.3 SEQ ID NO:398 840GATCTGTGACATTTAAAAAT −11.8 −15.7 51 −3.9 0 −5 SEQ ID NO:399 841TGATCTGTGACATTTAAAAA −11.8 −15.7 50.9 −3.9 0 −5 SEQ ID NO:400 29GGTGGTCTATQCTTTAGTCC −11.7 −26 78.2 −14.3 0 −3.9 SEQ ID NO:401 87GGTTCCCTGCTGGAGGCTCC −11.7 −32.6 89.7 −19.7 −1.1 −8 SEQ ID NO:402 106CTTGAAGAATATAATGGAAG −11.7 −14.6 48.5 −2.9 0 −2.7 SEQ ID NO:403 181CAGCATTAGTGGCAGCAACA −11.7 −24.6 70.8 −12 −0.8 −2.4 SEQ ID NO:404 189ATGGACATCAGCATTAGTGG −11.7 −22.5 67.2 −10.8 0 −4.1 SEQ ID NO:405 290TGCACTCACATTCTTGGCCG −11.7 −26.9 74.5 −14.7 0 −7.6 SEQ ID NO:406 750GTTTCCTGGAATCTTTCAGG −11.7 −23.6 70.2 −10.1 −1.8 −8.8 SEQ ID NO:407 871TTGGCAGACCCCATTTGAAG −11.7 −25.4 70.1 −13 −0.5 −4 SEQ ID NO:408 872ATTGGCAGACCCCATTTGAA −11.7 −25.4 69.8 −13 −0.5 −4 SEQ ID NO:409 873AATTGGCAGACCCCATTTGA −11.7 −25.4 69.8 −13 −0.5 −4 SEQ ID NO:410 996GAATGAGTGAAAGATGAACA −11.7 −16.3 51.8 −4.6 0 −2.9 SEQ ID NO:411 1005AGATTTGTCGAATGAGTGAA −11.7 −18.8 57.8 −6.2 −0.7 −5 SEQ ID NO:412 304CAGGAACCAATCTTTGCACT −11.6 −23.1 66 −11 −0.1 −7.8 SEQ ID NO:413 390TTCTTCACATTGCCCTTGAA −11.6 −24.3 69.4 −12.7 0 −3.5 SEQ ID NO:414 571AAGAAGAGTGTCTGGTAGGT −11.6 −22 67.7 −10.4 0 −2.9 SEQ ID NO:415 645GATCTTGAAAAACATGCTTT −11.6 −17.6 54.6 −6 0 −5 SEQ ID NO:416 724AGCCTAAGCCTGGGTAAGGG −11.6 −27.4 75.8 −14.4 −1.3 −8.2 SEQ ID NO:417 846AATTTTGATCTGTGACATTT −11.6 −18.4 57.9 −6.8 0 −4.9 SEQ ID NO:418 1008GAAAGATTTGTCGAATGAGT −11.6 −18.1 56 −5.6 −0.7 −5 SEQ ID NO:419 112AAGTTGCTTGAAGAATATAA −11.5 −15.9 51.5 −4.4 0 −2.9 SEQ ID NO:420 214CCCTGGATTCAGGCTGCTAG −11.5 −28.2 78.7 −14 −2.7 −9.6 SEQ ID NO:421 396CTTGTTTTCTTCACATTGCC −11.5 −23.8 70.8 −12.3 0 −3 SEQ ID NO:422 550TGCTCACTGTCTTCTTGGCT −11.5 −26.8 79.5 −14.8 0.1 −3.7 SEQ ID NO:423 908GCAGTGAATAGGGTAAAATG −11.5 −18.5 56.7 −7 0 −4.2 SEQ ID NO:424 127TGTCGGTGCAGCTGTAAGTT −11.4 −25.5 74.6 −13.4 0 −8.9 SEQ ID NO:425 182TCAGCATTAGTGGCAGCAAC −11.4 −24.3 71.3 −12 −0.8 −5.8 SEQ ID NO:426 276TGGCCGCCTTCCTGGAGCCA −11.4 −34.2 87.4 −19.2 −3.6 −10.7 SEQ ID NO:427 621GAGCACTGGAATGATTTAGG −11.4 −21 62.9 −9.6 0 −4.1 SEQ ID NO:428 709AAGGGGAGGGCACAGGCTAA −11.4 −26.2 73.4 −13.4 −1.3 −4 SEQ ID NO:429 749TTTCCTGGAATCTTTCAGGT −11.4 −23.6 70.2 −10.1 −2.1 −8.9 SEQ ID NO:430 851GAAACAATTTTGATCTGTGA −11.4 −17.4 54.7 −5.5 −0.2 −5.8 SEQ ID NO:431 921CTGGATTCAGTCTGCAGTGA −11.4 −24.7 73.9 −11.8 −0.5 −10.9 SEQ ID NO:432 997CGAATGAGTGAAAGATGAAC −11.4 −16.4 51.5 −5 0 −2 SEQ ID NO:433 68CTGATCCCTGGGGATGACTC −11.3 −27.1 75.9 −13.8 −1.4 −11.9 SEQ ID NO:434 277TTGGCCGCCTTCCTGGAGCC −11.3 −33.6 86.9 −19.8 −2.5 −10 SEQ ID NO:435 303AGGAACCAATCTTTGCACTC −11.3 −22.8 66.3 −11 −0.1 −7.8 SEQ ID NO:436 352CTTCTTTGGCAGCCCAGACA −11.3 −28.2 78.5 −15.8 −1 −8.1 SEQ ID NO:437 362AGGGGCACTGCTTCTTTGGC −11.3 −28.5 81.7 −16.5 −0.4 −6.3 SEQ ID NO:438 876GTGAATTGGCAGACCCCATT −11.3 −26.5 72.6 −14.5 −0.5 −4 SEQ ID NO:439 26GGTCTATGCTTTAGTCCCAG −11.2 −26.3 77.2 −14.6 −0.2 −4.6 SEQ ID NO:440 264TGGAGCCATCTCCTAGAAGC −11.2 −26.3 74.8 −11.9 −3.2 −8.6 SEQ ID NO:441 262GAGCCATCTCCTAGAAGCCT −11.1 −28 77.9 −15.9 −0.9 −5.6 SEQ ID NO:442 456TTGAGAAATTGCTGGCAGGC −11.1 −23.4 67.6 −11.5 −0.3 −9 SEQ ID NO:443 478AAAGCTTCTTAGCTGACATT −11.1 −20.8 62.9 −7.3 −2.4 −7 SEQ ID NO:444 705GGAGGGCACAGGCTAAGACT −11.1 −26.2 74.5 −14.4 −0.5 −4.3 SEQ ID NO:445 5CCAGCGTTCCCATTTGAGGG −11 −28.9 77.8 −16.8 −1 −9.2 SEQ ID NO:446 40ATACTCAGCCTGGTGGTCTA −11 −26.4 77.5 −14.8 −0.3 −4.8 SEQ ID NO:447 41GATACTCAGCCTGGTGGTCT −11 −27.3 79.6 −15.7 −0.3 −4.9 SEQ ID NO:448 180AGCATTAGTGGCAGCAACAG −11 −23.9 69.9 −12 −0.8 −2.4 SEQ ID NO:449 345GGCAGCCCAGACACTGTCAT −11 −29.1 80.5 −16.6 −1.4 −8.9 SEQ ID NO:450 357CACTGCTTCTTTGGCAGCCC −11 −29.6 81.9 −15.5 −3.1 −8.1 SEQ ID NO:451 446GCTGGCAGGCTCTGGAATGC −11 −28.5 80.1 −16.6 −0.7 −6 SEQ ID NO:452 490CAAAGGCAGAGCAAAGCTTC −11 −21.9 63.8 −9.9 −0.9 −7.7 SEQ ID NO:453 748TTCCTGGAATCTTTCAGGTA −11 −23.2 69.3 −10.1 −2.1 −8.9 SEQ ID NO:454 1007AAAGATTTGTCGAATGAGTG −11 −17.5 54.7 −5.6 −0.7 −5 SEQ ID NO:455 473TTCTTAGCTGACATTGTTTG −10.9 −20.9 64.6 −10 0 −5.1 SEQ ID NO:456 523TTTAATTGGAAGAGTGGGCG −10.9 −21.2 62.2 −10.3 0 −4 SEQ ID NO:457 720TAAGCCTGGGTAAGGGGAGG −10.9 −25.7 72.5 −13.4 −1.3 −4.9 SEQ ID NO:458 838TCTGTGACATTTAAAAATAT −10.9 −14.8 49.2 −3.9 0 −5 SEQ ID NO:459 839ATCTGTGACATTTAAAAATA −10.9 −14.8 49.2 −3.9 0 −5 SEQ ID NO:460 922TCTGGATTCAGTCTGCAGTG −10.9 −24.5 74.3 −11.8 −1.1 −11.7 SEQ ID NO:461 923ATCTGGATTCAGTCTGCAGT −10.9 −24.5 74.5 −11.8 −1.1 −11.7 SEQ ID NO:462 960CAGAGTGAGGGTCTTGGTGG −10.9 −25.7 76.6 −14.8 0 −2.6 SEQ ID NO:463 970CCAATGGAGACAGAGTGAGG −10.9 −22.9 66.5 −11.1 −0.8 −5.2 SEQ ID NO:464 1068AGACCGTGTCTGGTTCATTG −10.9 −25 72.7 −12.7 −1.3 −7.5 SEQ ID NO:465 1082TATTATCTTTAATAAGACCG −10.9 −16.6 52.7 −4.8 −0.7 −4.7 SEQ ID NO:466 32CCTGGTGGTCTATGCTTTAG −10.8 −25.3 74.5 −14.5 0 −3.6 SEQ ID NO:467 330GTCATGAATTTTCTTCTCGG −10.8 −21.6 65.2 −10.8 0.1 −6.7 SEQ ID NO:468 432GAATGCTTGTTTGGCTTTCT −10.8 −23.5 69.9 −11 −1.7 −5.4 SEQ ID NO:469 494CCTACAAAGGCAGAGCAAAG −10.8 −21.5 61.8 −9.8 −0.7 −4.6 SEQ ID NO:470 691AAGACTGACGAGAGAAGAAG −10.8 −17.6 54.4 −6.8 0 −3.5 SEQ ID NO:471 114GTAAGTTGCTTGAAGAATAT −10.7 −17.8 56.2 −7.1 0 −4.3 SEQ ID NO:472 263GGAGCCATCTCCTAGAAGCC −10.7 −28.3 78.6 −15.1 −2.5 −8.2 SEQ ID NO:473 358GCACTGCTTCTTTGGCAGCC −10.7 −29.4 82.9 −15.6 −3.1 −9.8 SEQ ID NO:474 371AATGATCACAGGGGCACTGC −10.7 −24.9 71 −12.7 −1.4 −8.4 SEQ ID NO:475 455TGAGAAATTGCTGGCAGGCT −10.7 −24.2 69.2 −12.3 −1.1 −7.5 SEQ ID NO:476 647ATGATCTTGAAAAACATGCT −10.7 −17.4 54 −6.7 0 −5 SEQ ID NO:477 755CTACAGTTTCCTGGAATCTT −10.7 −22.7 67.6 −10.6 −1.3 −4.6 SEQ ID NO:478 797TTTCCTGATTGCATTTAAGG −10.7 −21.1 63.2 −10.4 0 −5.1 SEQ ID NO:479 1006AAGATTTGTCGAATGAGTGA −10.7 −18.8 57.8 −7.2 −0.7 −5 SEQ ID NO:480 239CTCGGTCCCTGTGGCCTCTG −10.6 −32.2 86.9 −20 −1.5 −7.2 SEQ ID NO:481 267TCCTGGAGCCATCTCCTAGA −10.6 −28.5 80 −14.7 −3.2 −7.9 SEQ ID NO:482 291TTGCACTCACATTCTTGGCC −10.6 −26.2 75 −15.6 0 −6.2 SEQ ID NO:483 361GGGGCACTGCTTCTTTGGCA −10.6 −29.2 82.4 −17.3 −1.2 −7.2 SEQ ID NO:484 365CACAGGGGCACTGCTTCTTT −10.6 −27.1 77.5 −15 −1.4 −6.5 SEQ ID NO:485 519ATTGGAAGAGTGGGCGCTCA −10.6 −25.8 72.9 −12.5 −2.7 −10 SEQ ID NO:486 644ATCTTGAAAAACATGCTTTT −10.6 −17.1 53.7 −6 −0.2 −7.1 SEQ ID NO:487 856TGAAGGAAACAATTTTGATC −10.6 −15.8 51 −5.2 0 −5.8 SEQ ID NO:488 881TGGTTGTGAATTGGCAGACC −10.6 −24.2 69.9 −12.9 −0.4 −4.7 SEQ ID NO:489 147ATTAGAACTTTCATCGCAAC −10.5 −19.3 58.6 −8.8 0 −4.2 SEQ ID NO:490 346TGGCAGCCCAGACACTGTCA −10.5 −29.1 80.3 −16.6 −2 −9.6 SEQ ID NO:491 351TTCTTTGGCAGCCCAGACAC −10.5 −27.5 77.1 −16.1 −0.7 −8.1 SEQ ID NO:492 708AGGGGAGGGCACAGGCTAAG −10.5 −26.9 76.1 −15 −1.3 −4 SEQ ID NO:493 743GGAATCTTTCAGGTAATTAA −10.5 −18.2 57.1 −6.8 −0.8 −5.8 SEQ ID NO:494 760GGAAGCTACAGTTTCCTGGA −10.5 −24.9 72.3 −12.9 −1.4 −9.1 SEQ ID NO:495 1014ACCTCAGAAAGATTTGTCGA −10.5 −21.2 62.4 −9.8 −0.7 −4.8 SEQ ID NO:496 6GCCAGCGTTCCCATTTGAGG −10.4 −29.5 79.5 −19.1 0 −4.1 SEQ ID NO:497 39TACTCAGCCTGGTGGTCTAT −10.4 −26.4 77.5 −15.5 −0.2 −4.9 SEQ ID NO:498 72GCTCCTGATCCCTGGGGATG −10.4 −30.1 81.7 −17.7 −1.4 −11.9 SEQ ID NO:499 124CGGTGCAGCTGTAAGTTGCT −10.4 −26.6 75.8 −12.2 −4 −9.4 SEQ ID NO:500 574GAGAAGAAGAGTGTCTGGTA −10.4 −20.8 64.3 −10.4 0 −2.9 SEQ ID NO:501 728ATTAAGCCTAAGCCTGGGTA −10.4 −24.8 70.3 −14.4 0 −5.4 SEQ ID NO:502 10CCAGGCCAGCGTTCCCATTT −10.3 −31.6 82.7 −20.8 0 −7.7 SEQ ID NO:503 265CTGGAGCCATCTCCTAGAAG −10.3 −25.4 72.4 −11.9 −3.2 −7.4 SEQ ID NO:504 389TCTTCACATTGCCCTTGAAA −10.3 −23.5 66.9 −12.7 −0.2 −3.6 SEQ ID NO:505 746CCTGGAATCTTTCAGGTAAT −10.3 −22 65 10.1 −1.5 −7.7 SEQ ID NO:506 860CATTTGAAGGAAACAATTTT −10.3 −15.7 50.6 −5.4 0 −3.2 SEQ ID NO:507 493CTACAAAGGCAGAGCAAAGC −10.2 −21.3 62.1 −10.2 −0.7 −4.6 SEQ ID NO:508 548CTCACTGTCTTCTTGGCTGA −10.2 −25.6 76.2 −15.4 0 −3.7 SEQ ID NO:509 747TCCTGGAATCTTTCAGGTAA −10.2 −22.4 66.6 −10.1 −2.1 −8.9 SEQ ID NO:510 987AAAGATGAACAAGTAGGCCA −10.2 −19.9 58.8 −9.2 0 −7.7 SEQ ID NO:511 209GATTCAGGCTGCTAGAGACC −10.1 −25.5 74.3 −14.9 −0.1 −6.1 SEQ ID NO:512 356ACTGCTTCTTTGGCAGCCCA −10.1 −29.6 81.9 −16.4 −3.1 −8.1 SEQ ID NO:513 725AAGCCTAAGCCTGGGTAAGG −10.1 −25.5 71 −14.4 −0.9 −7.5 SEQ ID NO:514 764GCTAGGAAGCTACAGTTTCC −10.1 −24.6 72.5 −12.9 −1.5 −9.1 SEQ ID NO:515 855GAAGGAAACAATTTTGATCT −10.1 −16.7 52.9 −6.6 0 −5.8 SEQ ID NO:516 76GGAGGCTCCTGATCCCTGGG −10 −31.3 84.9 −20.5 −0.5 −8.6 SEQ ID NO:517 208ATTCAGGCTGCTAGAGACCA −10 −25.6 74 −14.9 −0.4 −6.1 SEQ ID NO:518 268TTCCTGGAGCCATCTCCTAG −10 −28 79 −15.5 −2.5 −7.3 SEQ ID NO:519 288CACTCACATTCTTGGCCGCC −10 −28.9 78.1 −18.4 0 −7.6 SEQ ID NO:520 344GCAGCCCAGACACTGTCATG −10 −27.9 77.7 −16.6 −1.2 −8.9 SEQ ID NO:521 354TGCTTCTTTGGCAGCCCAGA −10 −29.1 81 −18 −1 −8.1 SEQ ID NO:522 472TCTTAGCTGACATTGTTTGA −10 −21.4 65.6 −11.4 0 −5.4 SEQ ID NO:523 848ACAATTTTGATCTGTGACAT −10 −19.1 59 −9.1 0 −4.9 SEQ ID NO:524 880GGTTGTGAATTGGCAGACCC −10 −26.2 73.6 −15.5 −0.5 −4.1 SEQ ID NO:525 925GAATCTGGATTCAGTCTGCA −10 −23.2 69.4 −11.8 −1.1 −10.3 SEQ ID NO:526 146TTAGAACTTTCATCGCAACT −9.9 −20.2 60.4 −10.3 0 −4.2 SEQ ID NO:527 167GCAACAGGAGGAGGGAAGAG −9.9 −23.2 67.2 −13.3 0 −3.4 SEQ ID NO:528 355CTGCTTCTTTGGCAGCCCAG −9.9 −29.4 81.6 −17.3 −2.2 −7.9 SEQ ID NO:529 388CTTCACATTGCCCTTGAAAT −9.9 −23.1 65.4 −12.7 −0.2 −3.6 SEQ ID NO:530 692TAAGACTGACGAGAGAAGAA −9.9 −17.3 53.8 −7.4 0 −3.5 SEQ ID NO:531 693CTAAGACTGACGAGAGAAGA −9.9 −18.9 57.4 −9 0 −3.5 SEQ ID NO:532 757AGCTACAGTTTCCTGGAATC −9.9 −23.5 69.8 −12.9 −0.4 −8.3 SEQ ID NO:533 849AACAATTTTGATCTGTGACA −9.9 −18.4 57.1 −8 −0.2 −4.9 SEQ ID NO:534 866AGACCCCATTTGAAGGAAAC −9.9 −22.2 62.6 −12.3 0 −3.4 SEQ ID NO:535 1009AGAAAGATTTGTCGAATGAG −9.9 −16.9 53.4 −7 0.1 −5 SEQ ID NO:536 1098TTTTTTTTTAAACCTATATT −9.9 −15.7 51.6 −5.8 0 −4.4 SEQ ID NO:537 1099TTTTTTTTTTAAACCTATAT −9.9 −15.7 51.6 −5.8 0 −4.4 SEQ ID NO:538 212CTGGATTCAGGCTGCTAGAG −9.8 −24.8 73.1 −14.2 −0.6 −7.6 SEQ ID NO:539 235GTCCCTGTGGCCTCTGGCGA −9.8 −33.3 88.8 −21 −2.5 −7.7 SEQ ID NO:540 302GGAACCAATCTTTGCACTCA −9.8 −23.5 67.2 −13.2 −0.1 −5.1 SEQ ID NO:541 353GCTTCTTTGGCAGCCCAGAC −9.8 −29.3 81.9 −18.4 −1 −8.1 SEQ ID NO:542 556TAGGTGTGCTCACTGTCTTC −9.8 −25.2 77.4 −13.4 −2 −4.2 SEQ ID NO:543 600GTGGGTACAGTGGGAGAGTG −9.8 −25.4 76 −15.6 0 −5.2 SEQ ID NO:544 646TGATCTTGAAAAACATGCTT −9.8 −17.5 54.3 −7.7 0 −5 SEQ ID NO:545 785ATTTAAGGTTAAATGACACT −9.8 −16.6 53.1 −6.2 −0.3 −6.5 SEQ ID NO:546 920TGGATTCAGTCTGCAGTGAA −9.8 −23.1 69.3 −11.8 −0.5 −10.8 SEQ ID NO:547 13GTCCCAGGCCAGCGTTCCCA −9.7 −35 90.5 −24.8 0 −7.7 SEQ ID NO:548 35CAGCCTGGTGGTCTATGCTT −9.7 −28 80.4 −17.7 −0.3 −4.9 SEQ ID NO:549 73GGCTCCTGATCCCTGGGGAT −9.7 −31.3 84.5 −19.7 −1.2 −11.9 SEQ ID NO:550 123GGTGCAGCTGTAAGTTGCTT −9.7 −25.9 76.4 −12.2 −4 −11.4 SEQ ID NO:551 166CAACAGGAGGAGGGAAGAGA −9.7 −22 64.4 −12.3 0 0 SEQ ID NO:552 329TCATGAATTTTCTTCTCGGG −9.7 −21.6 64.6 −11.1 −0.6 −5.9 SEQ ID NO:553 552TGTGCTCACTGTCTTCTTGG −9.7 −25.3 76.2 −15.6 0 −5.5 SEQ ID NO:554 674AAGACACTAGAGAGAGCAAC −9.7 −19.4 59.5 −9.7 0 −4.5 SEQ ID NO:555 744TGGAATCTTTCAGGTAATTA −9.7 −18.9 59.1 −8.3 −0.8 −5.6 SEQ ID NO:556 915TCAGTCTGCAGTGAATAGGG −9.7 −23.3 70.1 −13 0 −8.4 SEQ ID NO:557 1083ATATTATCTTTAATAAGACC −9.7 −15.8 51.8 −4.8 −1.2 −5.2 SEQ ID NO:558 107GCTTGAAGAATATAATGGAA −9.6 −16.4 52.1 −6.8 0 −2.8 SEQ ID NO:559 305TCAGGAACCAATCTTTGCAC −9.6 −22.6 65.6 −12.5 −0.1 −7.8 SEQ ID NO:560 392TTTTCTTCACATTGCCCTTG −9.6 −24.6 71.1 −15 0 −3 SEQ ID NO:561 721CTAAGCCTGGGTAAGGGGAG −9.6 −25.4 71.9 −15 −0.6 −4.7 SEQ ID NO:562 850AAACAATTTTGATCTGTGAC −9.6 −17 54 −6.9 −0.2 −4.9 SEQ ID NO:563 1013CCTCAGAAAGATTTGTCGAA −9.6 −20.3 59.9 −9.8 −0.7 −5 SEQ ID NO:564 1015TACCTCAGAAAGATTTGTCG −9.6 −20.3 60.6 −9.8 −0.7 −3.2 SEQ ID NO:565 328CATGAATTTTCTTCTCGGGG −9.5 −22.4 65.7 −12.1 −0.6 −4.4 SEQ ID NO:566 752CAGTTTCCTGGAATCTTTCA −9.5 −23.1 68.7 −12.7 −0.8 −4.6 SEQ ID NO:567 924AATCTGGATTCAGTCTGCAG −9.5 −22.6 68.3 −11.8 −1.1 −9.9 SEQ ID NO:568 941GGGATAAGTATGTGTAGAAT −9.5 −18.9 59 −9.4 0 −1.8 SEQ ID NO:569 207TTCAGGCTGCTAGAGACCAT −9.4 −25.6 74 −14.9 −1.2 −6.7 SEQ ID NO:570 445CTGGCAGGCTCTGGAATGCT −9.4 −27.6 77.7 −16.6 −1.5 −6.7 SEQ ID NO:571 702GGGCACAGGCTAAGACTGAC −9.4 −25.2 72.1 −14.4 −1.3 −5.6 SEQ ID NO:572 875TGAATTGGCAGACCCCATTT −9.4 −25.4 69.8 −15.3 −0.5 −4 SEQ ID NO:573 33GCCTGGTGGTCTATGCTTTA −9.3 −27.1 78.8 −17.2 −0.3 −4.7 SEQ ID NO:574 240CCTCGGTCCCTGTGGCCTCT −9.3 −34.2 90.5 −23.3 −1.5 −7.2 SEQ ID NO:575 247AGCCTGGCCTCGGTCCCTGT −9.3 −34.7 91.5 −24.6 0 −9.2 SEQ ID NO:576 301GAACCAATCTTTGCACTCAC −9.3 −22.5 65.3 −13.2 0 −5 SEQ ID NO:577 377CCTTGAAATGATCACAGGGG −9.3 −22.4 64.2 −11.5 −1.6 −7.1 SEQ ID NO:578 787GCATTTAAGGTTAAATGACA −9.3 −18 55.8 −6 −2.7 −11 SEQ ID NO:579 986AAGATGAACAAGTAGGCCAA −9.3 −19.9 58.8 −10.1 0 −7.7 SEQ ID NO:580 61CTGGGGATGACTCAGGTCAG −9.2 −25.4 74.4 −13.8 −2.4 −6.6 SEQ ID NO:581 71CTCCTGATCCCTGGGGATGA −9.2 −28.9 78.7 −17.7 −1.4 −11.9 SEQ ID NO:582 84TCCCTGCTGGAGGCTCCTGA −9.2 −31.6 85.9 −21.1 −1.2 −7.1 SEQ ID NO:583 86GTTCCCTGCTGGAGGCTCCT −9.2 −32.3 89 −21.8 −1.2 −8 SEQ ID NO:584 116CTGTAAGTTGCTTGAAGAAT −9.2 −19 58.6 −9.8 0 −4.3 SEQ ID NO:585 477AAGCTTCTTAGCTGACATTG −9.2 −21.5 65 −9.9 −2.4 −7.1 SEQ ID NO:586 703AGGGCACAGGCTAAGACTGA −9.2 −25 71.7 −14.4 −1.3 −5.6 SEQ ID NO:587 704GAGGGCACAGGCTAAGACTG −9.2 −25 71.7 −14.4 −1.3 −5.3 SEQ ID NO:588 739TCTTTCAGGTAATTAAGCCT −9.2 −21.8 65.5 −12 −0.3 −5.4 SEQ ID NO:589 761AGGAAGCTACAGTTTCCTGG −9.2 −24.3 71.2 −12.9 −2.2 −10.6 SEQ ID NO:590 246GCCTGGCCTCGGTCCCTGTG −9.1 −34.7 90.8 −25.1 0 −8 SEQ ID NO:591 648AATGATCTTGAAAAACATGC −9.1 −15.8 50.6 −6.7 0 −5 SEQ ID NO:592 707GGGGAGGGCACAGGCTAAGA −9.1 −27.5 77.2 −17 −1.3 −4 SEQ ID NO:593 729AATTAAGCCTAAGCCTGGGT −9.1 −24.4 68.6 −14.4 −0.8 −5.4 SEQ ID NO:594 745CTGGAATCTTTCAGGTAATT −9.1 −20.1 61.6 −10.1 −0.8 −4.3 SEQ ID NO:595 11CCCAGGCCAGCGTTCCCATT −9 −33.5 85.5 −24 0 −7.7 SEQ ID NO:596 14AGTCCCAGGCCAGCGTTCCC −9 −34.3 90 −24.8 0 −7.7 SEQ ID NO:597 31CTGGTGGTCTATGCTTTAGT −9 −24.5 74.3 −15.5 0 −3.9 SEQ ID NO:598 190CATGGACATCAGCATTAGTG −9 22 65.8 −13 0 −4.1 SEQ ID NO:599 701GGCACAGGCTAAGACTGACG −9 −24.8 69.5 −14.4 −1.3 −5.4 SEQ ID NO:600 722CCTAAGCCTGGGTAAGGGGA −9 −27.4 75.1 −17 −1.3 −6.9 SEQ ID NO:601 753ACAGTTTCCTGGAATCTTTC −9 −22.6 68.1 −12.2 −1.3 −4.6 SEQ ID NO:602 38ACTCAGCCTGGTGGTCTATG −8.9 −26.7 77.9 −17.2 −0.3 −4.9 SEQ ID NO:603 70TCCTGATCCCTGGGGATGAC −8.9 −28.2 77.4 −17.7 −0.8 −11.3 SEQ ID NO:604 464GACATTGTTTGAGAAATTGC −8.9 −18.7 57.8 −9.8 0 −5.5 SEQ ID NO:605 673AGACACTAGAGAGAGCAACA −8.9 −20.8 62.8 −11.9 0 −4.1 SEQ ID NO:606 742GAATCTTTCAGGTAATTAAG −8.9 −17 54.7 −8.1 0 −5 SEQ ID NO:607 754TACAGTTTCCTGGAATCTTT −8.9 −21.9 66 −11.6 −1.3 −4.6 SEQ ID NO:608 861CCATTTGAAGGAAACAATTT −8.9 −17.6 53.8 −8.7 0 −3.2 SEQ ID NO:609 919GGATTCAGTCTGCAGTGAAT −8.9 −23.1 69.4 −11.8 −1.5 −12.8 SEQ ID NO:610 926AGAATCTGGATTCAGTCTGC −8.9 −22.5 68.5 −11.8 −1.7 −11 SEQ ID NO:611 995AATGAGTGAAAGATGAACAA −8.9 −15 49 −6.1 0 −2.5 SEQ ID NO:612 83CCCTGCTGGAGGCTCCTGAT −8.8 −31.2 84 −21.1 −1.2 −7.1 SEQ ID NO:613 211TGGATTCAGGCTGCTAGAGA −8.8 −24.5 72.4 −15.7 0 −6.6 SEQ ID NO:614 331TGTCATGAATTTTCTTCTCG −8.8 −20.4 62.5 −10.8 −0.6 −6.7 SEQ ID NO:615 386TCACATTGCCCTTGAAATGA −8.8 −22.7 64.4 −12.7 −1.1 −4.3 SEQ ID NO:616 643TCTTGAAAAACATGCTTTTT −8.8 −17.2 54 −7.5 −0.7 −8.5 SEQ ID NO:617 700GCACAGGCTAAGACTGACGA −8.8 −24.2 68.3 −14.4 −0.9 −5.4 SEQ ID NO:618 727TTAAGCCTAAGCCTGGGTAA −8.8 −24.1 68.1 −14.4 −0.8 −4.9 SEQ ID NO:619 740ATCTTTCAGGTAATTAAGCC −8.8 −20.9 63.5 −12.1 0 −5 SEQ ID NO:620 798CTTTCCTGATTGCATTTAAG −8.8 −20.8 62.5 −12 0 −5.1 SEQ ID NO:621 1075TTTAATAAGACCGTGTCTGG −8.8 −20.7 61.4 −10.5 −1.3 −8.3 SEQ ID NO:622 12TCCCAGGCCAGCGTTCCCAT −8.7 −33.8 86.9 −25.1 0 −6.9 SEQ ID NO:623 69CCTGATCCCTGGGGATGACT −8.7 −28.7 77.6 −18 −1.4 −11.9 SEQ ID NO:624 266CCTGGAGCCATCTCCTAGAA −8.7 −27.4 75.7 −15.5 −3.2 −7.7 SEQ ID NO:625 360GGGCACTGCTTCTTTGGCAG −8.7 −28 80.1 −17.3 −2 −9.7 SEQ ID NO:626 378CCCTTGAAATGATCACAGGG −8.7 −23.2 65.3 −11.5 −3 −7.9 SEQ ID NO:627 726TAAGCCTAAGCCTGGGTAAG −8.7 −24 68 −14.4 −0.8 −4.9 SEQ ID NO:628 759GAAGCTACAGTTTCCTGGAA −8.7 −23 67.3 −12.9 −1.3 −8.6 SEQ ID NO:629 867CAGACCCCATTTGAAGGAAA −8.7 −22.7 63.2 −14 0 −3.4 SEQ ID NO:630 1034TTTTGTCCCACCTCGCTCTT −8.7 −29 79.9 −20.3 0 −3.1 SEQ ID NO:631 1035TTTTTGTCCCACCTCGCTCT −8.7 −29 79.9 −20.3 0 −3.1 SEQ ID NO:632 348TTTGGCAGCCCAGACACTGT −8.6 −28.2 78.3 −18.5 −1 −9.1 SEQ ID NO:633 381TTGCCCTTGAAATGATCACA −8.6 −22.7 64.4 −13.4 −0.5 −6.8 SEQ ID NO:634 387TTCACATTGCCCTTGAAATG −8.6 −22.2 63.5 −12.7 −0.7 −4 SEQ ID NO:635 444TGGCAGGCTCTGGAATGCTT −8.6 −26.8 76.1 −16.6 −1.5 −6.7 SEQ ID NO:636 454GAGAAATTGCTGGCAGGCTC −8.6 −24.6 70.9 −14.8 −1.1 −7.5 SEQ ID NO:637 496CTCCTACAAAGGCAGAGCAA −8.6 −23.5 66.7 −13.7 −1.1 −6.3 SEQ ID NO:638 575GGAGAAGAAGAGTGTCTGGT −8.6 −22.3 67.6 −13.7 0 −2.9 SEQ ID NO:639 738CTTTCAGGTAATTAAGCCTA −8.6 −21.1 63.4 −12 −0.2 −5.3 SEQ ID NO:640 763CTAGGAAGCTACAGTTTCCT −8.6 −23.7 70.1 −12.9 −2.2 −10.7 SEQ ID NO:641 788TGCATTTAAGGTTAAATGAC −8.6 −17.3 54.6 −6 −2.7 −11 SEQ ID NO:642 929TGTAGAATCTGGATTCAGTC −8.6 −20.7 64.7 −10.3 −1.7 −11 SEQ ID NO:643 115TGTAAGTTGCTTGAAGAATA −8.5 −17.8 56.1 −9.3 0 −4.3 SEQ ID NO:644 119CAGCTGTAAGTTGCTTGAAG −8.5 −21.6 65 −12.2 −0.6 −8.8 SEQ ID NO:645 122GTGCAGCTGTAAGTTGCTTG −8.5 −24.7 73.5 −12.2 −4 −11.4 SEQ ID NO:646 350TCTTTGGCAGCCCAGACACT −8.5 −28.3 78.7 −18.7 −1 −7.7 SEQ ID NO:647 380TGCCCTTGAAATGATCACAG −8.5 −22.6 64.3 −13.4 −0.5 −6.8 SEQ ID NO:648 789TTGCATTTAAGGTTAAATGA −8.5 −17.2 54.4 −6 −2.7 −11 SEQ ID NO:649 1016TTACCTCAGAAAGATTTGTC −8.5 −19.6 60.4 −11.1 0 −2.5 SEQ ID NO:650 117GCTGTAAGTTGCTTGAAGAA −8.4 −20.8 62.7 −12.4 0 −4.3 SEQ ID NO:651 385CACATTGCCCTTGAAATGAT −8.4 −22.3 63.1 −12.7 −1.1 −4.3 SEQ ID NO:652 463ACATTGTTTGAGAAATTGCT −8.4 −19 58.5 −10.6 0 −4 SEQ ID NO:653 524GTTTAATTGGAAGAGTGGGC −8.4 −21.6 65 −13.2 0 −2.9 SEQ ID NO:654 622AGAGCACTGGAATGATTTAG −8.4 −19.8 60.5 −11.4 0 −4.1 SEQ ID NO:655 756GCTACAGTTTCCTGGAATCT −8.4 −24.4 71.6 −14.6 −1.3 −8.3 SEQ ID NO:656 786CATTTAAGGTTAAATGACAC −8.4 −16.4 52.5 −6 −2 −9.9 SEQ ID NO:657 25GTCTATGCTTTAGTCCCAGG −8.3 −26.3 77.2 −18 0 −3.9 SEQ ID NO:658 283ACATTCTTGGCCGCCTTCCT −8.3 −30.3 81.1 −21.5 0 −8 SEQ ID NO:659 300AACCAATCTTTGCACTCACA −8.3 −22.6 65.2 −14.3 0 −5 SEQ ID NO:660 349CTTTGGCAGCCCAGACACTG −8.3 −27.9 76.8 −18.5 −1 −8.5 SEQ ID NO:661 1033TTTGTCCCACCTCGCTCTTA −8.3 −28.6 79 −20.3 0 −3.1 SEQ ID NO:662 1043CTTTTTTTTTTTTGTCCCAC −8.3 −22.5 67.4 −14.2 0 −1.6 SEQ ID NO:663 287ACTCACATTCTTGGCCGCCT −8.2 −29.1 78.9 −20.4 0 −8 SEQ ID NO:664 332CTGTCATGAATTTTCTTCTC −8.2 −20.5 64.1 −11.5 −0.6 −6.7 SEQ ID NO:665 433GGAATGCTTGTTTGGCTTTC −8.2 −23.8 70.6 −13.9 −1.7 −5.4 SEQ ID NO:666 460TTGTTTGAGAAATTGCTGGC −8.2 −21.1 63.2 −12.9 0 −5.5 SEQ ID NO:667 510GTGGGCGCTCAGAGCTCCTA −8.2 −30.3 84.5 −20.8 1.5 −10.6 SEQ ID NO:668 511AGTGGGCGCTCAGAGCTCCT −8.2 −30.6 85.5 −21.1 1.5 −10.6 SEQ ID NO:669 1092TTTAAACCTATATTATCTTT −8.2 −16.3 52.9 −8.1 0 −4 SEQ ID NO:670 1093TTTTAAACCTATATTATCTT −8.2 −16.3 52.9 −8.1 0 −4.4 SEQ ID NO:671 108TGCTTGAAGAATATAATGGA −8.1 −17.1 53.7 −9 0 −3.6 SEQ ID NO:672 284CACATTCTTGGCCGCCTTCC −8.1 −30.1 80.2 −21.5 0 −8 SEQ ID NO:673 374TGAAATGATCACAGGGGCAC −8.1 −22.1 64.1 −13.4 −0.3 −6.3 SEQ ID NO:674 384ACATTGCCCTTGAAATGATC −8.1 −22 63.3 −12.7 −1.1 −4.3 SEQ ID NO:675 461ATTGTTTGAGAAATTGCTGG −8.1 −19.3 59.2 −11.2 0 −4 SEQ ID NO:676 624TGAGAGCACTGGAATGATTT −8.1 −20.7 62.1 −12.6 0 −3.4 SEQ ID NO:677 625TTGAGAGCACTGGAATGATT −8.1 −20.7 62.1 −12.6 0 −4.2 SEQ ID NO:678 758AAGCTACAGTTTCCTGGAAT −8.1 −22.4 66 −12.9 −1.3 −8.6 SEQ ID NO:679 928GTAGAATCTGGATTCAGTCT −8.1 −21.6 66.9 −11.7 −1.7 −11 SEQ ID NO:680 299ACCAATCTTTGCACTCACAT −8 −23.3 67.3 −15.3 0 −5 SEQ ID NO:681 572GAAGAAGAGTGTCTGGTAGG −8 −21.4 65.6 −13.4 0 −2.9 SEQ ID NO:682 120GCAGCTGTAAGTTGCTTGAA −7.9 −23.4 69 −12.2 −3.3 −11.4 SEQ ID NO:683 121TGCAGCTGTAAGTTGCTTGA −7.9 −24.1 71.3 −12.2 −4 −11.4 SEQ ID NO:684 359GGCACTGCTTCTTTGGCAGC −7.9 −28.6 82 −17.6 −3.1 −10.1 SEQ ID NO:685 375TTGAAATGATCACAGGGGCA −7.9 −22.6 3.9 13.4 −0.5 −6.8 SEQ ID NO:686 631TGCTTTTTGAGAGCACTGGA −7.9 −23.7 70 −13.8 −2 −5.9 SEQ ID NO:687 741AATCTTTCAGGTAATTAAGC −7.9 −18.2 57.5 −10.3 0 −5 SEQ ID NO:688 17TTTAGTCCCAGGCCAGCGTT −7.8 −29.8 81.7 −21.5 0 −7.7 SEQ ID NO:689 294TCTTTGCACTCACATTCTTG −7.8 −22.6 68.2 −14.8 0 −5 SEQ ID NO:690 295ATCTTTGCACTCACATTCTT −7.8 −22.6 68.4 −14.8 0 −4.7 SEQ ID NO:691 630GCTTTTTGAGAGCACTGGAA −7.8 −23 67.8 −13.8 −1.3 −4.6 SEQ ID NO:692 771GACACTAGCTAGGAAGCTAC −7.8 −22.4 66.9 −11.8 −2.8 −9.9 SEQ ID NO:693 780AGGTTAAATGACACTAGCTA −7.8 −19.5 59.8 −11.2 −0.1 −5.6 SEQ ID NO:694 1091TTAAACCTATATTATCTTTA −7.8 −15.9 52 −8.1 0 −2.3 SEQ ID NO:695 1097TTTTTTTTAAACCTATATTA −7.8 −15.3 50.7 −7.5 0 −4.1 SEQ ID NO:696 278CTTGGCCGCCTTCCTGGAGC −7.7 −32.5 85.5 −23.6 −0.8 −10 SEQ ID NO:697 306CTCAGGAACCAATCTTTGCA −7.7 −23.3 66.9 −15.6 0.2 −6.3 SEQ ID NO:698 335ACACTGTCATGAATTTTCTT −7.7 −19.9 61.5 −12.2 0 −6.7 SEQ ID NO:699 507GGCGCTCAGAGCTCCTACAA −7.7 −28.1 77.5 −19.8 2.3 −9.1 SEQ ID NO:700 599TGGGTACAGTGGGAGAGTGA −7.7 −24.8 73.7 −17.1 0 −5.2 SEQ ID NO:701 697CAGGCTAAGACTGACGAGAG −7.7 −22.1 64.4 −14.4 0 −4.9 SEQ ID NO:702 1074TTAATAAGACCGTGTCTGGT −7.7 −21.8 64.1 −12.7 −1.3 −8.3 SEQ ID NO:703 34AGCCTGGTGGTCTATGCTTT −7.6 −27.4 79.8 −19.2 −0.3 −4.9 SEQ ID NO:704 36TCAGCCTGGTGGTCTATGCT −7.6 −28.3 82 −20.1 −0.3 −4.9 SEQ ID NO:705 373GAAATGATCACAGGGGCACT −7.6 −23 66.1 −14.9 −0.2 −7 SEQ ID NO:706 449ATTGCTGGCAGGCTCTGGAA −7.6 −26.8 76.1 −18 −1.1 −7.5 SEQ ID NO:707 694GCTAAGACTGACGAGAGAAG −7.6 −20.1 60 −12.5 0 −3.5 SEQ ID NO:708 730TAATTAAGCCTAAGCCTGGG −7.6 −22.9 65.1 −14.4 −0.8 −5.8 SEQ ID NO:709 126GTCGGTGCAGCTGTAAGTTG −7.5 −25.5 74.6 −16.9 −1 −8.9 SEQ ID NO:710 184CATCAGCATTAGTGGCAGCA −7.5 −25.5 74.3 −18 0 −5.3 SEQ ID NO:711 437CTCTGGAATGCTTGTTTGGC −7.5 −24.5 71.7 −17 0 −3.6 SEQ ID NO:712 518TTGGAAGAGTGGGCGCTCAG −7.5 −25.8 73.2 −15.6 −2.7 −10.1 SEQ ID NO:713 762TAGGAAGCTACAGTTTCCTG −7.5 −22.8 67.9 −12.9 −2.4 −11.1 SEQ ID NO:714 879GTTGTGAATTGGCAGACCCC −7.5 −27 74.6 −18.8 −0.5 −4 SEQ ID NO:715 319TCTTCTCGGGGCTCTCAGGA −7.4 −28.4 82 −21 0 −4.1 SEQ ID NO:716 327ATGAATTTTCTTCTCGGGGC −7.4 −23.5 68.7 −15.3 −0.6 −4.1 SEQ ID NO:717 457TTTGAGAAATTGCTGGCAGG −7.4 −21.7 63.9 −13.6 0 −9 SEQ ID NO:718 629CTTTTTGAGAGCACTGGAAT −7.4 −21.2 63.5 −13.8 0 −4.2 SEQ ID NO:719 765AGCTAGGAAGCTACAGTTTC −7.4 −22.6 68.9 −12.9 −2.3 −7.8 SEQ ID NO:720 779GGTTAAATGACACTAGCTAG −7.4 −19.5 59.8 −11.2 −0.1 −9.5 SEQ ID NO:721 781AAGGTTAAATGACACTAGCT −7.4 −19.1 58.4 −11.2 −0.1 −5.1 SEQ ID NO:722 1084TATATTATCTTTAATAAGAC −7.4 −13.5 47.3 −4.8 −1.2 −5.2 SEQ ID NO:723 286CTCACATTCTTGGCCGCCTT −7.3 −29 78.7 −21.2 0 −8 SEQ ID NO:724 341GCCCAGACACTGTCATGAAT −7.3 −25.3 71 −17.3 −0.4 −7.1 SEQ ID NO:725 517TGGAAGAGTGGGCGCTCAGA −7.3 −26.3 74.2 −17.1 −1.9 −10.1 SEQ ID NO:726 672GACACTAGAGAGAGCAACAA −7.3 −20.1 60.5 −12.8 0 −4.5 SEQ ID NO:727 778GTTAAATGACACTAGCTAGG −7.3 −19.5 59.8 −11.2 0 −9.9 SEQ ID NO:728 791GATTGCATTTAAGGTTAAAT −7.3 −17.2 54.4 −9.3 −0.3 −6.5 SEQ ID NO:729 918GATTCAGTCTGCAGTGAATA −7.3 −21.6 66.1 −11.8 −1.7 −12.9 SEQ ID NO:730 191CCATGGACATCAGCATTAGT −7.2 −24 69.7 −16.8 0 −7.3 SEQ ID NO:731 347TTGGCAGCCCAGACACTGTC −7.2 −28.5 79.7 −20.1 −1.1 −8.7 SEQ ID NO:732 379GCCCTTGAAATGATCACAGG −7.2 −23.8 66.8 −14.9 −1.7 −6.8 SEQ ID NO:733 434TGGAATGCTTGTTTGGCTTT −7.2 −23.4 68.8 −15.3 −0.7 −4 SEQ ID NO:734 442GCAGGCTCTGGAATGCTTGT −7.2 −26.8 77 −18.5 −1 −6.7 SEQ ID NO:735 784TTTAAGGTTAAATGACACTA −7.2 −16.3 52.6 −8.6 −0.1 −4.7 SEQ ID NO:736 916TTCAGTCTGCAGTGAATAGG −7.2 −22.2 67.7 −13.9 −0.2 −10.2 SEQ ID NO:737 917ATTCAGTCTGCAGTGAATAG −7.2 −21 64.9 −11.8 −1.1 −12 SEQ ID NO:738 7GGCCAGCGTTCCCATTTGAG −7.1 −29.5 79.5 −22.4 0 −7 SEQ ID NO:739 495TCCTACAAAGGCAGAGCAAA −7.1 −21.9 62.9 −13.6 −1.1 −6.2 SEQ ID NO:740 626TTTGAGAGCACTGGAATGAT −7.1 −20.7 62.1 −13.6 0 −4.2 SEQ ID NO:741 751AGTTTCCTGGAATCTTTCAG −7.1 −22.4 67.8 −14.4 −0.8 −8.3 SEQ ID NO:742 884ATCTGGTTGTGAATTGGCAG −7.1 −22.7 67.7 −15.6 0 −4 SEQ ID NO:743 37CTCAGCCTGGTGGTCTATGC −7 −28.3 82 −20.7 −0.3 −4.9 SEQ ID NO:744 497GCTCCTACAAAGGCAGAGCA −7 −26 73.1 −16.6 −2.4 −7.9 SEQ ID NO:745 699CACAGGCTAAGACTGACGAG −7 −22.4 64.6 −14.4 −0.9 −5.4 SEQ ID NO:746 723GCCTAAGCCTGGGTAAGGGG −7 −28.6 78 −20.2 −1.3 −8.2 SEQ ID NO:747 772TGACACTAGCTAGGAAGCTA −7 −22.2 66.2 −12.4 −2.8 −9 SEQ ID NO:748 790ATTGCATTTAAGGTTAAATG −7 −16.6 53.1 −7.3 −2.3 −10.5 SEQ ID NO:749 1090TAAACCTATATTATCTTTAA −7 −15.1 50 −8.1 0 −2.2 SEQ ID NO:750 206TCAGGCTGCTAGAGACCATG −6.9 −25.5 73.5 −17.3 −1.2 −6.7 SEQ ID NO:751 320TTCTTCTCGGGGCTCTCAGG −6.9 −27.9 81 −21 0 −4.1 SEQ ID NO:752 698ACAGGCTAAGACTGACGAGA −6.9 −22.3 64.7 −14.4 −0.9 −5.4 SEQ ID NO:753 883TCTGGTTGTGAATTGGCAGA −6.9 −23.3 69.1 −15.7 −0.5 −4.2 SEQ ID NO:754 334CACTGTCATGAATTTTCTTC −6.8 −20.1 62.4 −13.3 0 −6.2 SEQ ID NO:755 448TTGCTGGCAGGCTCTGGAAT −6.8 −26.8 76.1 −18.8 −1.1 −7.5 SEQ ID NO:756 637AAAACATGCTTTTTGAGAGC −6.8 −18.9 57.8 −11.1 −0.9 −6.3 SEQ ID NO:757 767CTAGCTAGGAAGCTACAGTT −6.8 −22.7 68.3 −12.9 −3 −8.5 SEQ ID NO:758 59GGGGATGACTCAGGTCAGGA −6.7 −26.3 76.7 −17.7 −1.9 −6.1 SEQ ID NO:759 450AATTGCTGGCAGGCTCTGGA −6.7 −26.8 76.1 −18.9 −1.1 −7.5 SEQ ID NO:760 777TTAAATGACACTAGCTAGGA −6.7 −18.9 58.1 −11.2 0 −9.9 SEQ ID NO:761 30TGGTGGTCTATGCTTTAGTC −6.6 −24 74 −17.4 0 −3.9 SEQ ID NO:762 77TGGAGGCTCCTGATCCCTGG −6.6 −30.1 82.1 −22.2 −1.2 −7 SEQ ID NO:763 109TTGCTTGAAGAATATAATGG −6.6 −16.6 52.8 −10 0 −3.6 SEQ ID NO:764 376CTTGAAATGATCACAGGGGC −6.6 −22.2 64.6 −14.9 −0.5 −6.8 SEQ ID NO:765 436TCTGGAATGCTTGTTTGGCT −6.6 −24.5 71.7 −17 −0.7 −4 SEQ ID NO:766 770ACACTAGCTAGGAAGCTACA −6.6 −22.5 66.7 −12.9 −3 −9.9 SEQ ID NO:767 773ATGACACTAGCTAGGAAGCT −6.6 −22.5 66.7 −13.6 −2.3 −9.9 SEQ ID NO:768 1032TTGTCCCACCTCGCTCTTAC −6.6 −28.7 79.2 −22.1 0 −3.1 SEQ ID NO:769 799ACTTTCCTGATTGCATTTAA −6.5 −21 62.9 −14.5 0 −5.1 SEQ ID NO:770 854AAGGAAACAATTTTGATCTG −6.5 −16.1 51.6 −9.6 0 −5.8 SEQ ID NO:771 1010CAGAAAGATTTGTCGAATGA −6.5 −17.6 54.4 −10.2 −0.7 −5 SEQ ID NO:772 118AGCTGTAAGTTGCTTGAAGA −6.4 −21.5 65.1 −14.4 −0.5 −6.2 SEQ ID NO:773 326TGAATTTTCTTCTCGGGGCT −6.4 −24.4 70.7 −17.2 −0.6 −4.3 SEQ ID NO:774 336GACACTGTCATGAATTTTCT −6.4 −20.4 62.5 −13.3 −0.4 −6.9 SEQ ID NO:775 382ATTGCCCTTGAAATGATCAC −6.4 −22 63.3 −14.9 −0.5 −6.8 SEQ ID NO:776 465TGACATTGTTTGAGAAATTG −6.4 −16.9 53.8 −10.5 0 −5.5 SEQ ID NO:777 471CTTAGCTGACATTGTTTGAG −6.4 −21 64.3 −14.6 0 −5.4 SEQ ID NO:778 1073TAATAAGACCGTGTCTGGTT −6.4 −21.8 64.1 −14 −1.3 −7.8 SEQ ID NO:779 186GACATCAGCATTAGTGGCAG −6.3 −23.8 70.6 −16.6 −0.8 −4.1 SEQ ID NO:780 241GCCTCGGTCCCTGTGGCCTC −6.3 −35.1 93.1 −26.8 −2 −7.2 SEQ ID NO:781 261AGCCATCTCCTAGAAGCCTG −6.3 −27.4 76.4 −20.1 −0.9 −4.3 SEQ ID NO:782 318CTTCTCGGGGCTCTCAGGAA −6.3 −27.3 77.4 −21 0 −4.1 SEQ ID NO:783 627TTTTGAGAGCACTGGAATGA −6.3 −20.8 62.5 −14.5 0 −4.2 SEQ ID NO:784 737TTTCAGGTAATTAAGCCTAA −6.3 −19.5 59.4 −12.5 −0.4 −5.5 SEQ ID NO:785 1085CTATATTATCTTTAATAAGA −6.3 −14.2 48.7 −6.8 −1 −5.2 SEQ ID NO:786 298CCAATCTTTGCACTCACATT −6.2 −23.2 67.1 −17 0 −5 SEQ ID NO:787 462CATTGTTTGAGAAATTGCTG −6.2 −18.8 57.9 −12.6 0 −4 SEQ ID NO:788 623GAGAGCACTGGAATGATTTA −6.2 −20.4 61.6 −14.2 0 −4.2 SEQ ID NO:789 766TAGCTAGGAAGCTACAGTTT −6.2 −21.9 66.6 −12.9 −2.8 −8.3 SEQ ID NO:790 833GACATTTAAAAATATTTATT −6.2 −12.3 44.2 −5.4 −0.4 −6.7 SEQ ID NO:791 1096TTTTTTTAAACCTATATTAT −6.2 −15.2 50.4 −9 0 −4.4 SEQ ID NO:792 42GGATACTCAGCCTGGTGGTC −6.1 −27.6 80.2 −20.9 −0.3 −4.9 SEQ ID NO:793 245CCTGGCCTCGGTCCCTGTGG −6.1 −34.1 88.9 −28 −0.3 −7.2 SEQ ID NO:794 909TGCAGTGAATAGGGTAAAAT −6.1 −18.5 56.7 −12.4 0 −4.7 SEQ ID NO:795 942GGGGATAAGTATGTGTAGAA −6.1 −20.1 61.7 −14 0 −1.8 SEQ ID NO:796 1042TTTTTTTTTTTTGTCCCACC −6.1 −23.6 69.2 −17.5 0 −1.7 SEQ ID NO:797 16TTAGTCCCAGGCCAGCGTTC −6 −30.1 83.1 −23.6 0 −7.7 SEQ ID NO:798 506GCGCTCAGAGCTCCTACAAA −6 −26.2 72.6 −18.7 −1.4 −9.6 SEQ ID NO:799 642CTTGAAAAACATGCTTTTTG −6 −16.8 52.8 −9.2 −1.5 −9.1 SEQ ID NO:800 649AAATGATCTTGAAAAACATG −6 −13.3 45.6 −7.3 0 −4.9 SEQ ID NO:801 816ATTGACTTCTGTTTGCTACT −6 −22.1 67.4 −16.1 0 −3.6 SEQ ID NO:802 834TGACATTTAAAAATATTTAT −6 −12.2 43.9 −5.5 −0.4 −6.7 SEQ ID NO:803 836TGTGACATTTAAAAATATTT −6 −13.7 46.9 −7.7 0 −6.4 SEQ ID NO:804 439GGCTCTGGAATGCTTGTTTG −5.9 −24.5 71.7 −17.9 −0.5 −4 SEQ ID NO:805 441CAGGCTCTGGAATGCTTGTT −5.9 −25.1 72.9 −18.5 −0.5 −5.4 SEQ ID NO:806 776TAAATGACACTAGCTAGGAA −5.9 −18.1 55.9 −11.2 0 −9.9 SEQ ID NO:807 783TTAAGGTTAAATGACACTAG −5.9 −16.2 52.4 −10.3 0.7 −4 SEQ ID NO:808 1072AATAAGACCGTGTCTGGTTC −5.9 −22.5 66.1 −15.2 −1.3 −8.3 SEQ ID NO:809 85TTCCCTGCTGGAGGCTCCTG −5.8 −31.1 85 −24 −1.2 −8 SEQ ID NO:810 321TTTCTTCTCGGGGCTCTCAG −5.8 −26.8 78.7 −21 0 −4.1 SEQ ID NO:811 829TTTAAAAATATTTATTGACT −5.8 −12.5 44.7 −6 −0.4 −6.2 SEQ ID NO:812 248AAGCCTGGCCTCGGTCCCTG −5.7 −32.8 85.1 −26.3 0 −9.2 SEQ ID NO:813 323ATTTTCTTCTCGGGGCTCTC −5.7 −26.2 77.5 −20.5 0 −4.1 SEQ ID NO:814 325GAATTTTCTTCTCGGGGCTC −5.7 −24.8 72.5 −19.1 0 −3.9 SEQ ID NO:815 466CTGACATTGTTTGAGAAATT −5.7 −17.8 55.8 −12.1 0 −5.5 SEQ ID NO:816 800TACTTTCCTGATTGCATTTA −5.7 −21.4 64.4 −15.7 0 −5.1 SEQ ID NO:817 830ATTTAAAAATATTTATTGAC −5.7 −11.6 42.9 −5.2 −0.4 −6.7 SEQ ID NO:818 210GGATTCAGGCTGCTAGAGAC −5.6 −24.7 73.2 −19.1 0 −6.1 SEQ ID NO:819 638AAAAACATGCTTTTTGAGAG −5.6 −16.4 52.2 −9.8 −0.9 −8.3 SEQ ID NO:820 1039TTTTTTTTTGTCCCACCTCG −5.6 −25.4 71.7 −19.8 0 −2.4 SEQ ID NO:821 24TCTATGCTTTAGTCCCAGGC −5.5 −26.9 78.1 −21.4 0 −3.6 SEQ ID NO:822 183ATCAGCATTAGTGGCAGCAA −5.5 −24.1 70.6 −17.7 −0.8 −5.3 SEQ ID NO:823 185ACATCAGCATTAGTGGCAGC −5.5 −25 73.8 −18.6 −0.8 −4.7 SEQ ID NO:824 202GCTGCTAGAGACCATGGACA −5.5 −25.9 73.4 −19.7 0 −8.8 SEQ ID NO:825 296AATCTTTGCACTCACATTCT −5.5 −21.8 65.6 −16.3 0 −5 SEQ ID NO:826 525TGTTTAATTGGAAGAGTGGG −5.5 −19.8 60.7 −14.3 0 −2.6 SEQ ID NO:827 547TCACTGTCTTCTTGGCTGAG −5.5 −24.7 74.4 −19.2 0 −4.2 SEQ ID NO:828 632ATGCTTTTTGAGAGCACTGG −5.5 −23.1 68.6 −15.2 −2.4 −6.7 SEQ ID NO:829 768ACTAGCTAGGAAGCTACAGT −5.5 −22.8 68.5 −14.3 −3 −9.9 SEQ ID NO:830 835GTGACATTTAAAAATATTTA −5.5 −13.4 46.4 −7.4 −0.2 −6.7 SEQ ID NO:831 279TCTTGGCCGCCTTCCTGGAG −5.4 −31.1 83.1 −24.6 −0.3 −10 SEQ ID NO:832 534GGCTGAGAATGTTTAATTGG −5.4 −20.1 60.9 −14.7 0 −3.7 SEQ ID NO:833 576GGGAGAAGAAGAGTGTCTGG −5.4 −22.3 67 −16.9 0 −2.9 SEQ ID NO:834 636AAACATGCTTTTTGAGAGCA −5.4 −20.3 61 −13.2 −1.7 −5.9 SEQ ID NO:835 911TCTGCAGTGAATAGGGTAAA −5.4 −20.5 61.9 −14.5 0 −8.6 SEQ ID NO:836 1031TGTCCCACCTCGCTCTTACC −5.4 −30.6 82.2 −25.2 0 −3.1 SEQ ID NO:837 60TGGGGATGACTCAGGTCAGG −5.3 −25.7 75.1 −18 −2.4 −6.6 SEQ ID NO:838 769CACTAGCTAGGAAGCTACAG −5.3 −22.3 66.4 −14 −3 −9.9 SEQ ID NO:839 910CTGCAGTGAATAGGGTAAAA −5.3 −19.4 58.6 −14.1 0 −7.4 SEQ ID NO:840 1041TTTTTTTTTTTGTCCCACCT −5.3 −24.4 70.8 −19.1 0 −1.7 SEQ ID NO:841 342AGCCCAGACACTGTCATGAA −5.2 −25.3 71.3 −18.8 −1.2 −7.6 SEQ ID NO:842 503CTCAGAGCTCCTACAAAGGC −5.2 −24.8 71.3 −18.4 −1.1 −8.4 SEQ ID NO:843 792TGATTGCATTTAAGGTTAAA −5.2 −17.2 54.4 −12 0 −5.3 SEQ ID NO:844 793CTGATTGCATTTAAGGTTAA −5.2 −18.8 58.1 −13.6 0 −4.8 SEQ ID NO:845 440AGGCTCTGGAATGCTTGTTT −5.1 −24.5 72.1 −18.7 −0.5 −4 SEQ ID NO:846 443GGCAGGCTCTGGAATGCTTG −5.1 −26.8 76.1 −20.1 −1.5 −6.7 SEQ ID NO:847 501CAGAGCTCCTACAAAGGCAG −5.1 −24.2 69.2 −17.9 −1.1 −8.4 SEQ ID NO:848 826AAAAATATTTATTGACTTCT −5.1 −14 47.7 −8.9 0 −6.7 SEQ ID NO:849 58GGGATGACTCAGGTCAGGAT −5 −25.1 73.9 −17.7 −2.4 −6.6 SEQ ID NO:850 201CTGCTAGAGACCATGGACAT −5 −24.1 69.2 −18.4 0 −8.8 SEQ ID NO:851 340CCCAGACACTGTCATGAATT −5 −23.6 67.2 −17.3 −1.2 −7.6 SEQ ID NO:852 467GCTGACATTGTTTGAGAAAT −5 −19.5 59.4 −14.5 0 −5.5 SEQ ID NO:853 468AGCTGACATTGTTTGAGAAA −5 −19.5 59.6 −14.5 0 −4.9 SEQ ID NO:854 695GGCTAAGACTGACGAGAGAA −5 −21.3 62.2 −16.3 0 −3.7 SEQ ID NO:855 15TAGTCCCAGGCCAGCGTTCC −4.9 −32 86.2 −26.6 0 −7.7 SEQ ID NO:856 435CTGGAATGCTTGTTTGGCTT −4.9 −24.2 70.4 −18.4 −0.7 −4 SEQ ID NO:857 509TGGGCGCTCAGAGCTCCTAC −4.9 −29.3 81.5 −23.1 −1.5 −10.6 SEQ ID NO:858 512GAGTGGGCGCTCAGAGCTCC −4.9 −30.3 84.9 −23.1 −1.9 −12.4 SEQ ID NO:859 706GGGAGGGCACAGGCTAAGAC −4.9 −26.5 75.2 −20.2 −1.3 −4 SEQ ID NO:860 1011TCAGAAAGATTTGTCGAATG −4.9 −17.4 54.4 −11.6 −0.7 −5 SEQ ID NO:861 1040TTTTTTTTTTGTCCCACCTC −4.9 −24.7 72.1 −19.8 0 −1.7 SEQ ID NO:862 828TTAAAAATATTTATTGACTT −4.8 −12.5 44.7 −7 −0.4 −6.7 SEQ ID NO:863 458GTTTGAGAAATTGCTGGCAG −4.7 −21.7 64.4 −15.9 0 −10.1 SEQ ID NO:864 546CACTGTCTTCTTGGCTGAGA −4.7 −24.9 74.1 −20.2 0 −6 SEQ ID NO:865 774AATGACACTAGCTAGGAAGC −4.7 −20.9 62.6 −14.6 −1.5 −9.9 SEQ ID NO:866 1020GCTCTTACCTCAGAAAGATT −4.7 −21.9 65.1 −16.5 −0.4 −3.6 SEQ ID NO:867 1030GTCCCACCTCGCTCTTACCT −4.7 −31.5 84.3 −26.8 0 −3.1 SEQ ID NO:868 1038TTTTTTTTGTCCCACCTCGC −4.7 −27.1 75.5 −22.4 0 −2.7 SEQ ID NO:869 256TCTCCTAGAAGCCTGGCCTC −4.6 −29.2 81.4 −23.5 0 −10.1 SEQ ID NO:870 322TTTTCTTCTCGGGGCTCTCA −4.6 −26.9 78.7 −22.3 0 −4.1 SEQ ID NO:871 324AATTTTCTTCTCGGGGCTCT −4.6 −25.1 73.1 −20.5 0 −4.1 SEQ ID NO:872 200TGCTAGAGACCATGGACATC −4.5 −23.6 68.8 −18.4 0 −8.8 SEQ ID NO:873 650AAAATGATCTTGAAAAACAT −4.5 −12.6 44.2 −8.1 0 −4.2 SEQ ID NO:874 671ACACTAGAGAGAGCAACAAA −4.5 −18.8 57.3 −14.3 0 −4.5 SEQ ID NO:875 736TTCAGGTAATTAAGCCTAAG −4.5 −19.4 59.3 −14.2 −0.4 −5.5 SEQ ID NO:876 977AAGTAGGCCAATGGAGACAG −4.5 −22.5 65.4 −17.1 −0.8 −8.4 SEQ ID NO:877 18CTTTAGTCCCAGGCCAGCGT −4.4 −30.6 83.2 −25.7 0 −7.7 SEQ ID NO:878 333ACTGTCATGAATTTTCTTCT −4.4 −20.3 63.1 −15.1 −0.6 −6.7 SEQ ID NO:879 337AGACACTGTCATGAATTTTC −4.4 −19.5 60.7 −13.8 −1.2 −7.6 SEQ ID NO:880 500AGAGCTCCTACAAAGGCAGA −4.4 −24.1 69.3 −18.5 −1.1 −8.4 SEQ ID NO:881 514AAGAGTGGGCGCTCAGAGCT −4.4 −27.2 77.1 −20.1 −2.7 −9.6 SEQ ID NO:882 598GGGTACAGTGGGAGAGTGAG −4.4 −24.8 74.3 −20.4 0 −5.2 SEQ ID NO:883 43AGGATACTCAGCCTGGTGGT −4.3 −27.2 78.7 −21.8 −1 −6.7 SEQ ID NO:884 438GCTCTGGAATGCTTGTTTGG −4.3 −24.5 71.7 −20.2 0 −3.6 SEQ ID NO:885 628TTTTTGAGAGCACTGGAATG −4.3 −20.3 61.5 −16 0 −4.2 SEQ ID NO:886 639GAAAAACATGCTTTTTGAGA −4.3 −17 53.3 −11.1 −1.5 −9.1 SEQ ID NO:887 731GTAATTAAGCCTAAGCCTGG −4.3 −22.9 65.6 −17.7 −0.8 −6.5 SEQ ID NO:888 257ATCTCCTAGAAGCCTGGCCT −4.2 −28.8 79.5 −23.5 0 −10.1 SEQ ID NO:889 260GCCATCTCCTAGAAGCCTGG −4.2 −28.6 78.6 −23.7 −0.5 −4.2 SEQ ID NO:890 292TTTGCACTCACATTCTTGGC −4.2 −24.3 71.7 −20.1 0 −5 SEQ ID NO:891 505CGCTCAGAGCTCCTACAAAG −4.2 −24.4 68.8 −18.7 −1.4 −9.6 SEQ ID NO:892 535TGGCTGAGAATGTTTAATTG −4.2 −18.9 58.3 −14.7 0 −3.7 SEQ ID NO:893 827TAAAAATATTTATTGACTTC −4.2 −12.8 45.4 −8.1 0.1 −6.7 SEQ ID NO:894 1086CCTATATTATCTTTAATAAG −4.2 −15.6 51.3 −10.5 −0.8 −3.3 SEQ ID NO:895 199GCTAGAGACCATGGACATCA −4.1 −24.3 70.1 −19.5 0 −8.8 SEQ ID NO:896 383CATTGCCCTTGAAATGATCA −4.1 −22.5 63.9 −17.8 −0.3 −6.5 SEQ ID NO:897 451AAATTGCTGGCAGGCTCTGG −4.1 −25.5 72.3 −20.7 −0.5 −7.5 SEQ ID NO:898 499GAGCTCCTACAAAGGCAGAG −4.1 −24.1 69.3 −18.8 −1.1 −7.2 SEQ ID NO:899 515GAAGAGTGGGCGCTCAGAGC −4.1 −26.9 76.4 −20.1 −2.7 −10.1 SEQ ID NO:900 498AGCTCCTACAAAGGCAGAGC −4 −25.3 72.3 −19.2 −2.1 −7.1 SEQ ID NO:901 125TCGGTGCAGCTGTAAGTTGC −3.9 −26.1 75.5 −19.7 −2.5 −9.4 SEQ ID NO:902 205CAGGCTGCTAGAGACCATGG −3.9 −26.3 74.4 −21.1 −1.2 −8.3 SEQ ID NO:903 285TCACATTCTTGGCCGCCTTC −3.9 −28.5 78.5 −24.1 0 −8 SEQ ID NO:904 1037TTTTTTTGTCCCACCTCGCT −3.9 −27.9 77 −24 0 −3.1 SEQ ID NO:905 23CTATGCTTTAGTCCCAGGCC −3.8 −28.5 79.9 −24.7 0 −6.4 SEQ ID NO:906 502TCAGAGCTCCTACAAAGGCA −3.8 −24.6 70.5 −19.6 −1.1 −8.4 SEQ ID NO:907 459TGTTTGAGAAATTGCTGGCA −3.7 −21.7 64.1 −17.4 0 −8.4 SEQ ID NO:908 696AGGCTAAGACTGACGAGAGA −3.7 −22 64.5 −18.3 0 −3.7 SEQ ID NO:909 837CTGTGACATTTAAAAATATT −3.7 −14.5 48.4 −10.8 0 −5 SEQ ID NO:910 1021CGCTCTTACCTCAGAAAGAT −3.7 −22.6 65 −18.2 −0.4 −3.6 SEQ ID NO:911 78CTGGAGGCTCCTGATCCCTG −3.6 −29.8 81.5 −24.9 −1.2 −7 SEQ ID NO:912 508GGGCGCTCAGAGCTCCTACA −3.6 −30 82.7 −25.5 1.5 −9.9 SEQ ID NO:913 825AAAATATTTATTGACTTCTG −3.6 −14.7 49.3 −11.1 0 −6.7 SEQ ID NO:914 1012CTCAGAAAGATTTGTCGAAT −3.6 −18.3 56.3 −13.8 −0.7 −5 SEQ ID NO:915 1094TTTTTAAACCTATATTATCT −3.6 −16.3 52.9 −12.7 0 −4.4 SEQ ID NO:916 1095TTTTTTAAACCTATATTATC −3.6 −15.5 51.3 −11.9 0 −4.4 SEQ ID NO:917 57GGATGACTCAGGTCAGGATA −3.5 −23.6 70.5 −17.7 −2.4 −6.6 SEQ ID NO:918 81CTGCTGGAGGCTCCTGATCC −3.5 −29.6 82.4 −24.9 −1.1 −6.3 SEQ ID NO:919 293CTTTGCACTCACATTCTTGG −3.5 −23.4 69.3 −19.9 0 −5 SEQ ID NO:920 536TTGGCTGAGAATGTTTAATT −3.5 −19 58.7 −15.5 0 −3.7 SEQ ID NO:921 82CCTGCTGGAGGCTCCTGATC −3.4 −29.6 82.4 −24.9 −1.2 −7.1 SEQ ID NO:922 249GAAGCCTGGCCTCGGTCCCT −3.4 −33.4 86.6 −29.2 0 −9.2 SEQ ID NO:923 635AACATGCTTTTTGAGAGCAC −3.4 −21.2 63.6 −15.4 −2.4 −6.7 SEQ ID NO:924 832ACATTTAAAAATATTTATTG −3.4 −11.7 43 −7.6 −0.4 −6.7 SEQ ID NO:925 927TAGAATCTGGATTCAGTCTG −3.4 −20.4 63.4 −15.2 −1.7 −11 SEQ ID NO:926 309GCTCTCAGGAACCAATCTTT −3.3 −23.9 69.4 −20.1 −0.1 −4.6 SEQ ID NO:927 372AAATGATCACAGGGGCACTG −3.3 −22.4 64.7 −17.8 −1.2 −8.5 SEQ ID NO:928 447TGCTGGCAGGCTCTGGAATG −3.3 −26.7 75.5 −22.2 −1.1 −7 SEQ ID NO:929 526ATGTTTAATTGGAAGAGTGG −3.3 −18.6 58.1 −15.3 0 −2.9 SEQ ID NO:930 192ACCATGGACATCAGCATTAG −3.2 −23 67 −19.1 0 −8.8 SEQ ID NO:931 244CTGGCCTCGGTCCCTGTGGC −3.2 −33.9 90.1 −28.3 −2.4 −7.2 SEQ ID NO:932 343CAGCCCAGACACTGTCATGA −3.2 −26.7 74.7 −22.2 −1.2 −7.6 SEQ ID NO:933 782TAAGGTTAAATGACACTAGC −3.2 −17.9 56 −14.2 −0.1 −4.5 SEQ ID NO:934 824AAATATTTATTGACTTCTGT −3.2 −16.6 53.9 −13.4 0 −5.8 SEQ ID NO:935 339CCAGACACTGTCATGAATTT −3.1 −21.7 64 −17.3 −1.2 −7.6 SEQ ID NO:936 823AATATTTATTGACTTCTGTT −3.1 −17.4 56.1 −14.3 0 −3.8 SEQ ID NO:937 651CAAAATGATCTTGAAAAACA −3 −13.3 45.4 −10.3 0 −4.9 SEQ ID NO:938 504GCTCAGAGCTCCTACAAAGG −2.9 −24.8 71.3 −20.1 −1.8 −10.2 SEQ ID NO:939 19GCTTTAGTCCCAGGCCAGCG −2.8 −31.2 84 −27.9 −0.2 −7.7 SEQ ID NO:940 670CACTAGAGAGAGCAACAAAC −2.8 −18.8 57.3 −16 0 −4.5 SEQ ID NO:941 735TCAGGTAATTAAGCCTAAGC −2.8 −21.1 63 −17.6 −0.4 −5.5 SEQ ID NO:942 45TCAGGATACTCAGCCTGGTG −2.6 −25.9 75.3 −21.1 −2.2 −6.6 SEQ ID NO:943 577TGGGAGAAGAAGAGTGTCTG −2.6 −21.1 64.2 −18.5 0 −2.9 SEQ ID NO:944 453AGAAATTGCTGGCAGGCTCT −2.5 −24.9 71.5 −21.2 −1.1 −7.5 SEQ ID NO:945 1028CCCACCTCGCTCTTACCTCA −2.5 −31 81.8 −28.5 0 −3.1 SEQ ID NO:946 1087ACCTATATTATCTTTAATAA −2.5 −15.8 51.7 −12.7 −0.3 −3.3 SEQ ID NO:947 313CGGGGCTCTCAGGAACCAAT −2.4 −26.8 72.7 −23.4 −0.9 −4.6 SEQ ID NO:948 802GCTACTTTCCTGATTGCATT −2.4 −24.3 70.9 −21.9 0 −5.1 SEQ ID NO:949 312GGGGCTCTCAGGAACCAATC −2.3 −26.4 74.4 −23.1 −0.9 −4.6 SEQ ID NO:950 811CTTCTGTTTGCTACTTTCCT −2.3 −24.7 73.6 −22.4 0 −3.6 SEQ ID NO:951 1019CTCTTACCTCAGAAAGATTT −2.3 −20.2 61.3 −17.2 −0.4 −3.6 SEQ ID NO:952 46GTCAGGATACTCAGCCTGGT −2.2 −27.1 79.2 −22.7 −2.2 −6.6 SEQ ID NO:953 307TCTCAGGAACCAATCTTTGC −2.1 −23 67.3 −20.4 −0.1 −4.1 SEQ ID NO:954 280TTCTTGGCCGCCTTCCTGGA −2 −31.2 83.1 −28.1 −0.3 −10 SEQ ID NO:955 338CAGACACTGTCATGAATTTT −2 −19.8 60.5 −16.5 −1.2 −7.6 SEQ ID NO:956 633CATGCTTTTTGAGAGCACTG −2 −22.6 67.1 −18.2 −2.4 −6.7 SEQ ID NO:957 663GAGAGCAACAAACAAAATGA −2 −15.9 50.2 −13.9 0 −4.1 SEQ ID NO:958 665GAGAGAGCAACAAACAAAAT −2 −15.9 50.4 −13.9 0 −4.1 SEQ ID NO:959 666AGAGAGAGCAACAAACAAAA −2 −15.9 50.5 −13.9 0 −4.1 SEQ ID NO:960 813GACTTCTGTTTGCTACTTTC −2 −22.6 69.7 −20.6 0 −3.6 SEQ ID NO:961 55ATGACTCAGGTCAGGATACT −1.9 −22.9 69.1 −18.1 −2.9 −7.2 SEQ ID NO:962 259CCATCTCCTAGAAGCCTGGC −1.9 −28.6 78.6 −26 0 −8.8 SEQ ID NO:963 530GAGAATGTTTAATTGGAAGA −1.9 −16.7 53.4 −14.8 0 −2.9 SEQ ID NO:964 775AAATGACACTAGCTAGGAAG −1.9 −18.4 56.7 −15.5 0 −9.9 SEQ ID NO:965 831CATTTAAAAATATTTATTGA −1.9 −12.1 43.7 −9.5 −0.4 −6.7 SEQ ID NO:966 801CTACTTTCCTGATTGCATTT −1.8 −22.6 67 −20.8 0 −5.1 SEQ ID NO:967 80TGCTGGAGGCTCCTGATCCC −1.7 −30.7 83.9 −27.7 −1.2 −7 SEQ ID NO:968 203GGCTGCTAGAGACCATGGAC −1.7 −26.4 74.9 −23.9 −0.4 −8.8 SEQ ID NO:969 314TCGGGGCTCTCAGGAACCAA −1.7 −27.2 74.3 −24.5 −0.9 −4.6 SEQ ID NO:970 1017CTTACCTCAGAAAGATTTGT −1.7 −20.1 60.9 −18.4 0 −2.5 SEQ ID NO:971 242GGCCTCGGTCCCTGTGGCCT −1.6 −35.9 93.6 −30.1 −4.2 −10.8 SEQ ID NO:972 21ATGCTTTAGTCCCAGGCCAG −1.5 −28.6 79.9 −26.6 0 −7.7 SEQ ID NO:973 805TTTGCTACTTTCCTGATTGC −1.5 −23.7 70 −22.2 0 −3.6 SEQ ID NO:974 281ATTCTTGGCCGCCTTCCTGG −1.4 −30.6 81.8 −28.2 0 −10 SEQ ID NO:975 597GGTACAGTGGGAGAGTGAGG −1.4 −24.8 74.3 −23.4 0 −5.2 SEQ ID NO:976 662AGAGCAACAAACAAAATGAT −1.4 −15.3 49.1 −13.9 0 −4.1 SEQ ID NO:977 664AGAGAGCAACAAACAAAATG −1.4 −15.3 49.2 −13.9 0 −3.3 SEQ ID NO:978 732GGTAATTAAGCCTAAGCCTG −1.4 −22.9 65.6 −20.6 −0.8 −6.5 SEQ ID NO:979 812ACTTCTGTTTGCTACTTTCC −1.4 −24 72.2 −22.6 0 −3.6 SEQ ID NO:980 529AGAATGTTTAATTGGAAGAG −1.3 −16.1 52.3 −14.8 0 −2.9 SEQ ID NO:981 593CAGTGGGAGAGTGAGGTGGG −1.3 −26.1 76.8 −24.8 0 −3.1 SEQ ID NO:982 1022TCGCTCTTACCTCAGAAAGA −1.3 −23 66.5 −21.2 −0.2 −3.5 SEQ ID NO:983 1036TTTTTTGTCCCACCTCGCTC −1.3 −28.2 78.4 −26.9 0 −3.1 SEQ ID NO:984 315CTCGGGGCTCTCAGGAACCA −1.2 −28.8 78.5 −26.6 −0.9 −4.6 SEQ ID NO:985 44CAGGATACTCAGCCTGGTGG −1.1 −26.7 76.2 −24 −1.6 −6.7 SEQ ID NO:986 79GCTGGAGGCTCCTGATCCCT −1.1 −31.6 86.1 −29.2 −1.2 −7 SEQ ID NO:987 1029TCCCACCTCGCTCTTACCTC −1.1 −30.7 82.6 −29.6 0 −3.1 SEQ ID NO:988 255CTCCTAGAAGCCTGGCCTCG −1 −29.6 79.2 −27.7 −0.3 −9.5 SEQ ID NO:989 310GGCTCTCAGGAACCAATCTT −1 −25 71.6 −23.5 −0.1 −4.6 SEQ ID NO:990 578GTGGGAGAAGAAGAGTGTCT −1 −22.3 67.6 −21.3 0 −2.9 SEQ ID NO:991 1088AACCTATATTATCTTTAATA −1 −15.8 51.7 −14 −0.6 −3.1 SEQ ID NO:992 282CATTCTTGGCCGCCTTCCTG −0.9 −30.1 80.3 −28.7 0 −8 SEQ ID NO:993 452GAAATTGCTGGCAGGCTCTG −0.9 −24.9 71.1 −22.8 −1.1 −7.2 SEQ ID NO:994 533GCTGAGAATGTTTAATTGGA −0.9 −19.5 59.6 −18.6 0 −2.9 SEQ ID NO:995 804TTGCTACTTTCCTGATTGCA −0.9 −24.3 70.8 −22.9 −0.2 −4.8 SEQ ID NO:996 20TGCTTTAGTCCCAGGCCAGC −0.8 −30.4 84.5 −29.1 0 −7.7 SEQ ID NO:997 470TTAGCTGACATTGTTTGAGA −0.8 −20.7 63.6 −19.9 0 −5.4 SEQ ID NO:998 542GTCTTCTTGGCTGAGAATGT −0.7 −23.6 71.1 −22 −0.8 −8.1 SEQ ID NO:999 661GAGCAACAAACAAAATGATC −0.7 −15.7 50 −15 0 −4.1 SEQ ID NO:1000 810TTCTGTTTGCTACTTTCCTG −0.7 −23.8 71.4 −23.1 0 −3.6 SEQ ID NO:1001 198CTAGAGACCATGGACATCAG −0.6 −22.5 66.1 −21.2 0 −8.8 SEQ ID NO:1002 308CTCTCAGGAACCAATCTTTG −0.6 −22.1 65.1 −21 0.1 −4.6 SEQ ID NO:1003 669ACTAGAGAGAGCAACAAACA −0.6 −18.8 57.3 −18.2 0 −4.5 SEQ ID NO:1004 803TGCTACTTTCCTGATTGCAT −0.6 −24.2 70.4 −23.1 −0.2 −5.1 SEQ ID NO:1005 814TGACTTCTGTTTGCTACTTT −0.6 −22.2 67.8 −21.6 0 −3.6 SEQ ID NO:1006 56GATGACTCAGGTCAGGATAC −0.5 −22.6 68.4 −19.7 −2.4 −6.6 SEQ ID NO:1007 818TTATTGACTTCTGTTTGCTA −0.5 −20.8 64.5 −20.3 0 −3.6 SEQ ID NO:1008 1023CTCGCTCTTACCTCAGAAAG −0.5 −23.3 67.1 −22.8 0 −3.1 SEQ ID NO:1009 311GGGCTCTCAGGAACCAATCT −0.4 −26.1 73.8 −25.2 −0.1 −4.6 SEQ ID NO:1010 532CTGAGAATGTTTAATTGGAA −0.3 −17 53.8 −16.7 0 −2.9 SEQ ID NO:1011 806GTTTGCTACTTTCCTGATTG −0.2 −23.1 69 −22.9 0 −3.6 SEQ ID NO:1012 1089AAACCTATATTATCTTTAAT −0.2 −15.4 50.5 −15.2 0 −2.5 SEQ ID NO:1013 54TGACTCAGGTCAGGATACTC −0.1 −23.3 70.8 −20.5 −2.7 −6.8 SEQ ID NO:1014 808CTGTTTGCTACTTTCCTGAT −0.1 −23.9 70.7 −23.8 0 −3.6 SEQ ID NO:1015 596GTACAGTGGGAGAGTGAGGT 0 −24.8 75.2 −24.8 0 −4.6 SEQ ID NO:1016 654AAACAAAATGATCTTGAAAA 0 −11.9 42.9 −11.9 0 −5 SEQ ID NO:1017 297CAATCTTTGCACTCACATTC 0.1 −21.6 64.9 −21.7 0 −5 SEQ ID NO:1018 469TAGCTGACATTGTTTGAGAA 0.1 −19.9 61.1 −20 0 −5.3 SEQ ID NO:1019 819TTTATTGACTTCTGTTTGCT 0.2 −21.2 65.5 −21.4 0 −3.6 SEQ ID NO:1020 53GACTCAGGTCAGGATACTCA 0.3 −24 72.2 −22.2 −2.1 −5.1 SEQ ID NO:1021 516GGAAGAGTGGGCGCTCAGAG 0.3 −26.3 74.7 −23.9 −2.7 −10.1 SEQ ID NO:1022 531TGAGAATGTTTAATTGGAAG 0.3 −16.1 52.1 −16.4 0 −2.9 SEQ ID NO:1023 655CAAACAAAATGATCTTGAAA 0.3 −13.3 45.4 −13.6 0 −5 SEQ ID NO:1024 815TTGACTTCTGTTTGCTACTT 0.3 −22.2 67.8 −22.5 0 −3.6 SEQ ID NO:1025 1018TCTTACCTCAGAAAGATTTG 0.3 −19.3 59.3 −19.1 −0.2 −3.5 SEQ ID NO:1026 537CTTGGCTGAGAATGTTTAAT 0.4 −19.8 60.3 −20.2 0 −4 SEQ ID NO:1027 541TCTTCTTGGCTGAGAATGTT 0.4 −22.5 68 −22 −0.8 −8.1 SEQ ID NO:1028 317TTCTCGGGGCTCTCAGGAAC 0.5 −26.6 76.1 −27.1 0 −4.1 SEQ ID NO:1029 204AGGCTGCTAGAGACCATGGA 0.6 −26.2 74.6 −25.5 −1.2 −8.8 SEQ ID NO:1030 251TAGAAGCCTGGCCTCGGTCC 0.6 −30.2 81.3 −29.9 −0.3 −9.5 SEQ ID NO:1031 668CTAGAGAGAGCAACAAACAA 0.8 −17.9 55 −18.7 0 −4.1 SEQ ID NO:1032 316TCTCGGGGCTCTCAGGAACC 0.9 −28.5 79.3 −29.4 0 −3.3 SEQ ID NO:1033 809TCTGTTTGCTACTTTCCTGA 0.9 −24.3 72.4 −25.2 0 −3.6 SEQ ID NO:1034 528GAATGTTTAATTGGAAGAGT 1 −17.3 55 −18.3 0 −2.9 SEQ ID NO:1035 538TCTTGGCTGAGAATGTTTAA 1 −20.2 61.7 −21.2 0 −5.8 SEQ ID NO:1036 652ACAAAATGATCTTGAAAAAC 1 −12.8 44.6 −13.8 0 −5 SEQ ID NO:1037 653AACAAAATGATCTTGAAAAA 1.1 −11.9 42.9 −13 0 −5 SEQ ID NO:1038 660AGCAACAAACAAAATGATCT 1.1 −16 50.6 −17.1 0 −4.9 SEQ ID NO:1039 807TGTTTGCTACTTTCCTGATT 1.2 −23.1 69 −24.3 0 −3.4 SEQ ID NO:1040 250AGAAGCCTGGCCTCGGTCCC 1.4 −32.5 85.2 −33 −0.3 −9.5 SEQ ID NO:1041 822ATATTTATTGACTTCTGTTT 1.4 −18.2 58.5 −19.6 0 −2.5 SEQ ID NO:1042 47GGTCAGGATACTCAGCCTGG 1.6 −27.1 78.2 −26.5 −2.2 −7 SEQ ID NO:1043 539TTCTTGGCTGAGAATGTTTA 1.6 −21 64.2 −21.8 −0.6 −7.8 SEQ ID NO:1044 50TCAGGTCAGGATACTCAGCC 1.7 −26.1 76.9 −26.7 −1 −4.6 SEQ ID NO:1045 820ATTTATTGACTTCTGTTTGC 1.7 −20.3 63.4 −22 0 −2.6 SEQ ID NO:1046 258CATCTCCTAGAAGCCTGGCC 1.8 −28.6 78.6 −29.3 0 −10.1 SEQ ID NO:1047 656ACAAACAAAATGATCTTGAA 1.8 −14.2 47.2 −16 0 −5 SEQ ID NO:1048 49CAGGTCAGGATACTCAGCCT 1.9 −26.6 77.1 −26.7 −1.8 −4.9 SEQ ID NO:1049 243TGGCCTCGGTCCCTGTGGCC 1.9 −35 91.4 −32.8 −4.1 −10.6 SEQ ID NO:1050 513AGAGTGGGCGCTCAGAGCTC 1.9 −28.3 81.6 −27.5 −2.7 −12.3 SEQ ID NO:1051 579GGTGGGAGAAGAAGAGTGTC 2 −22.6 68.3 −24.6 0 −1.8 SEQ ID NO:1052 817TATTGACTTCTGTTTGCTAC 2 −20.9 64.7 −22.9 0 −3.6 SEQ ID NO:1053 527AATGTTTAATTGGAAGAGTG 2.1 −16.7 53.6 −18.8 0 −2.9 SEQ ID NO:1054 545ACTGTCTTCTTGGCTGAGAA 2.2 −23.5 70.3 −24.9 −0.6 −7.8 SEQ ID NO:1055 52ACTCAGGTCAGGATACTCAG 2.4 −23.4 71.1 −24.9 −0.8 −3.8 SEQ ID NO:1056 197TAGAGACCATGGACATCAGC 2.4 −23.4 68.4 −25.1 0 −8.8 SEQ ID NO:1057 1024CCTCGCTCTTACCTCAGAAA 2.4 −25.3 70.4 −27.7 0 −3.1 SEQ ID NO:1058 821TATTTATTGACTTCTGTTTG 2.5 −18.2 58.4 −20.7 0 −2.5 SEQ ID NO:1059 51CTCAGGTCAGGATACTCAGC 2.6 −25 75.1 −26.7 −0.8 −4.2 SEQ ID NO:1060 544CTGTCTTCTTGGCTGAGAAT 2.6 −23.3 69.7 −25.1 −0.6 −7.9 SEQ ID NO:1061 641TTGAAAAACATGCTTTTTGA 2.6 −16.5 52.2 −17.5 −1.5 −9.1 SEQ ID NO:1062 657AACAAACAAAATGATCTTGA 2.6 −14.2 47.2 −16.8 0 −5 SEQ ID NO:1063 1026CACCTCGCTCTTACCTCAGA 2.6 −27.6 76.7 −30.2 0 −3.1 SEQ ID NO:1064 634ACATGCTTTTTGAGAGCACT 2.8 −22.8 67.8 −23.2 −2.4 −6.7 SEQ ID NO:1065 1025ACCTCGCTCTTACCTCAGAA 3.2 −26.2 73.2 −29.4 0 −2.7 SEQ ID NO:1066 733AGGTAATTAAGCCTAAGCCT 3.4 −22.9 66 −25.4 −0.8 −6.6 SEQ ID NO:1067 196AGAGACCATGGACATCAGCA 3.5 −24.4 70.1 −27.3 0 −8.5 SEQ ID NO:1068 640TGAAAAACATGCTTTTTGAG 3.5 −16.4 52.1 −18.3 −1.5 −9.1 SEQ ID NO:1069 658CAACAAACAAAATGATCTTG 3.5 −14.3 47.3 −17.8 0 −4.9 SEQ ID NO:1070 667TAGAGAGAGCAACAAACAAA 3.8 −16.3 51.6 −20.1 0 −4.1 SEQ ID NO:1071 734CAGGTAATTAAGCCTAAGCC 3.8 −22.7 65.2 −25.6 −0.8 −6.8 SEQ ID NO:1072 1027CCACCTCGCTCTTACCTCAG 3.8 −29 78.8 −32.8 0 −3.1 SEQ ID NO:1073 543TGTCTTCTTGGCTGAGAATG 3.9 −22.4 67.5 −25.4 −0.8 −8.1 SEQ ID NO:1074 580AGGTGGGAGAAGAAGAGTGT 4 −22.2 67 −26.2 0 0 SEQ ID NO:1075 587GAGAGTGAGGTGGGAGAAGA 4 −22.9 68.7 −26.9 0 0 SEQ ID NO:1076 254TCCTAGAAGCCTGGCCTCGG 4.1 −29.9 79.8 −33.4 0.2 −8.7 SEQ ID NO:1077 253CCTAGAAGCCTGGCCTCGGT 4.3 −30.7 81.4 −34.1 −0.3 −9.5 SEQ ID NO:1078 540CTTCTTGGCTGAGAATGTTT 4.3 −22.2 66.8 −25.6 −0.8 −8.1 SEQ ID NO:1079 592AGTGGGAGAGTGAGGTGGGA 4.3 −26 77.1 −30.3 0 0 SEQ ID NO:1080 595TACAGTGGGAGAGTGAGGTG 4.5 −23.6 71.3 −28.1 0 −4.6 SEQ ID NO:1081 193GACCATGGACATCAGCATTA 4.7 −23.6 68.1 −27.6 0 −8.8 SEQ ID NO:1082 194AGACCATGGACATCAGCATT 4.9 −23.9 68.9 −28.1 0 −8.8 SEQ ID NO:1083 581GAGGTGGGAGAAGAAGAGTG 5.3 −21.6 65 −26.9 0 0 SEQ ID NO:1084 586AGAGTGAGGTGGGAGAAGAA 5.3 −21.6 65 −26.9 0 0 SEQ ID NO:1085 252CTAGAAGCCTGGCCTCGGTC 5.6 −29.1 79.8 −33.8 −0.3 −9.5 SEQ ID NO:1086 22TATGCTTTAGTCCCAGGCCA 5.7 −28.3 79 −33.5 0 −7.7 SEQ ID NO:1087 589GGGAGAGTGAGGTGGGAGAA 5.8 −24.7 72.5 −30.5 0 0 SEQ ID NO:1088 590TGGGAGAGTGAGGTGGGAGA 6.1 −25.4 74.8 −31.5 0 0 SEQ ID NO:1089 195GAGACCATGGACATCAGCAT 6.2 −24.4 69.8 −29.9 0 −8.8 SEQ ID NO:1090 594ACAGTGGGAGAGTGAGGTGG 6.4 −25.1 74.7 −31.5 0 −4.6 SEQ ID NO:1091 588GGAGAGTGAGGTGGGAGAAG 7 −23.5 70 −30.5 0 0 SEQ ID NO:1092 591GTGGGAGAGTGAGGTGGGAG 7.3 −26 77.1 −33.3 0 0 SEQ ID NO:1093 659GCAACAAACAAAATGATCTT 9 −16.1 50.7 −25.1 0 −4.9 SEQ ID NO:1094 582TGAGGTGGGAGAAGAAGAGT 9.4 −21.6 65 −31 0 −0.1 SEQ ID NO:1095 48AGGTCAGGATACTCAGCCTG 9.5 −25.9 75.8 −33.6 −1.8 −7 SEQ ID NO:1096 584AGTGAGGTGGGAGAAGAAGA 9.6 −21.6 65 −31.2 0 0 SEQ ID NO:1097 583GTGAGGTGGGAGAAGAAGAG 11.4 −21.6 65 −33 0 0 SEQ ID NO:1098 585GAGTGAGGTGGGAGAAGAAG 11.9 −21.6 65 −33.5 0 0 SEQ ID NO:1099

Example 15 Western Blot Analysis of VCC-1 Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to VCC-1 is used, with aradiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1. An antisense compound 8 to 30 nucleobases in length targeted to anucleic acid molecule encoding VCC-1, wherein said antisense compoundspecifically hybridizes with and inhibits the expression of VCC-1. 2.The antisense compound of claim 1 which is an antisense oligonucleotide.3. The antisense oligonucleotide of claim 2 comprising a nucleic acidsequence selected from the group consisting of at least eight contiguousbases of SEQ ID NO: 1-SEQ ID NO:
 1099. 4. The antisense oligonucleotideof claim 2 comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1-SEQ ID NO:
 1099. 5. The antisense compound ofclaim 2, wherein the antisense oligonucleotide comprises at least onemodified internucleoside linkage.
 6. The antisense compound of claim 5wherein the modified internucleoside linkage is a phosphorothioatelinkage.
 7. The antisense compound of claim 2, wherein the antisenseoligonucleotide comprises at least one modified sugar moiety.
 8. Theantisense compound of claim 7 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 9. The antisense compound of claim 2,wherein the antisense oligonucleotide comprises at least one modifiednucleobase.
 10. The antisense compound of claim 9 wherein the modifiednucleobase is a 5-methylcytosine.
 11. The antisense compound of claim 2,wherein the antisense oligonucleotide is a chimeric oligonucleotide. 12.A composition comprising the antisense compound of claim 1 and apharmaceutically acceptable carrier or diluent.
 13. The composition ofclaim 12 further comprising a colloidal dispersion system.
 14. Thecomposition of claim 13 wherein the antisense compound is an antisenseoligonucleotide.
 15. A method of inhibiting the expression of VCC-1 incells or tissues comprising contacting said cells or tissues with theantisense compound of claim 1 so that expression of VCC-1 is inhibited.16. A method of treating a human having a disease or conditionassociated with VCC-1 comprising administering to said animal atherapeutically or prophylactically effective amount of the antisensecompound of claim 1 so that expression of VCC-1 is inhibited.
 17. Themethod of claim 16 wherein the disease or condition is selected from thegroup consisting of diabetes, an immunological disorder, acardiovascular disorder, a neurologic disorder, an ischemia/reperfusioninjury, any form of cancer, and an angiogenic disorder. 18-21.(canceled)
 22. The method of claim 16 wherein the disease or conditionis any form of cancer.
 23. The method of claim 16 wherein the disease orcondition is an angiogenic disorder.